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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV210153	3/11	Homo sapiens	22Rv1	(d)(U)C Filtration DG	Allelein, Susann	2021	100%
Study summary Full title Potential and challenges of specifically isolating extracellular vesicles from heterogeneous populations All authors Susann Allelein, Paula Medina-Perez, Ana Leonor Heitor Lopes, Sabrina Rau, Gerd Hause, Andreas Kölsch, Dirk Kuhlmeier Journal Sci Rep Abstract Extracellular vesicles (EVs) have attracted interest due to their ability to provide diagnostic info (show more...)Extracellular vesicles (EVs) have attracted interest due to their ability to provide diagnostic information from liquid biopsies. Cells constantly release vesicles divers in size, content and features depending on the biogenesis, origin and function. This heterogeneity adds a layer of complexity when attempting to isolate and characterize EVs resulting in various protocols. Their high abundance in all bodily fluids and their stable source of origin dependent biomarkers make EVs a powerful tool in biomarker discovery and diagnostics. However, applications are limited by the quality of samples definition. Here, we compared frequently used isolation techniques: ultracentrifugation, density gradient centrifugation, ultrafiltration and size exclusion chromatography. Then, we aimed for a tissue-specific isolation of prostate-derived EVs from cell culture supernatants with immunomagnetic beads. Quality and quantity of EVs were confirmed by nanoparticle tracking analysis, western blot and electron microscopy. Additionally, a spotted antibody microarray was developed to characterize EV sub-populations. Current analysis of 16 samples on one microarray for 6 different EV surface markers in triplicate could be easily extended allowing a faster and more economical method to characterize samples. (hide) EV-METRIC 100% (99th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Filtration DG Protein markers EV: TSG101/ Alix/ CD9 non-EV: Calnexin Proteomics no EV density (g/ml) 1.08-1.11 Show all info Study aim New methodological development/Biomarker/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells 22Rv1 EV-harvesting Medium Serum free medium Cell viability (%) 95 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Pelleting performed No Density gradient Only used for validation of main results Yes Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5 Highest density fraction 40 Total gradient volume, incl. sample (mL) 16 Sample volume (mL) 0.5 Orientation Top-down Rotor type Surespin 630 (17 ml) Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 8.25 Pelleting: duration (min) 180 Pelleting: rotor type Surespin 630 (36 ml) Pelleting: speed (g) 100000 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method Fluorometric assay (e.g. Qubit, NanoOrange,...) Western Blot Detected EV-associated proteins CD9/ TSG101/ Alix Not detected contaminants Calnexin Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 140 EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 1.40E+11 EM EM-type Transmission electron microscopy Image type Close-up, Wide-field

	EV210118	1/4	Bos taurus	cheese manufacturing byproducts	DG tangential flow filtration Filtration	Sukreet, Sonal	2021	100%
Study summary Full title Isolation of extracellular vesicles from byproducts of cheesemaking by tangential flow filtration yields heterogeneous fractions of nanoparticles All authors Sonal Sukreet, Camila Pereira Braga, Thuy T. An, Jiri Adamec, Juan Cui, Benjamin Trible, Janos Zempleni Journal Journal of Dairy Science Abstract Extracellular vesicles (EV) in milk, particularly exosomes, have attracted considerable attention as (show more...)Extracellular vesicles (EV) in milk, particularly exosomes, have attracted considerable attention as bioactive food compounds and for their use in drug delivery. The utility of small EV in milk (sMEV) as an animal feed additive and in drug delivery would be enhanced by cost-effective large-scale protocols for the enrichment of sMEV from byproducts in dairy plants. Here, we tested the hypothesis that sMEV may be enriched from byproducts of cheesemaking by tangential flow filtration (EV-FF) and that the sMEV have properties similar to sMEV prepared by ultracentrifugation (sMEV-UC). Three fractions of EV were purified from the whey fraction of cottage cheese making by using EV-FF that passed through a membrane with a 50-kDa cutoff (50 penetrate; 50P), and subfractions of 50P that were retained (100 retentate; 100R) or passed through (100 penetrate; 100P) a membrane with a 100-kDa cutoff; sMEV-UC controls were prepared by serial ultracentrifugation. The abundance of sMEV (<200 nm) was less than 0.3% in EV-FF compared with sMEV-UC (1012/mL of milk). Despite the low EV count, the protein content (mg/mL) of 100R (63 ± 0.02; ± standard deviation) was higher than that of 50P (0.75 ± 0.10), 100P (0.65 ± 0.40), and sMEV-UC (27 ± 0.02). There were 17, 14, 35, and 75 distinct proteins detected by nontargeted mass spectrometry analysis in 50P, 100R, 100P, and sMEV-UC, respectively. Exosome markers CD9, CD63, CD81, HSP-70, PDCD6IP, and TSG101 were detected in control sMEV-UC but not in EV-FF by using targeted mass spectrometry and immunoblot analyses. Negative exosome markers, APOB, β-integrin, and histone H3 were below the limit of detection in EV-FF and control sMEV-UC analyzed by immunoblotting. The abundance of the major milk fat globule protein butyrophilin showed the following pattern: 100R ≫ 100P = 50P > sMEV-UC. More than 100 mature microRNA were detected in sMEV-UC by using sequencing analysis, compared with 36 to 60 microRNA in EV-FF. Only 100R and sMEV-UC yielded mRNA in quantities and qualities sufficient for sequencing analysis; an average of 276,000 and 838,000 reads were mapped to approximately 14,600 and 18,500 genes in 100R and sMEV-UC, respectively. In principal component analysis, microRNA, mRNA, and protein in EV-FF preparations clustered separately from control sMEV-UC. We conclude that under the conditions used here, flow filtration yields a heterogeneous population of milk EV. (hide) EV-METRIC 100% (50th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type cheese manufacturing byproducts Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture DG tangential flow filtration Filtration Protein markers EV: TSG101/ CD63/ CD81/ Alix/ HSP70/ CD9 non-EV: Integrin-beta/ Histone H3/ ApoB Proteomics yes EV density (g/ml) 1.255 Show all info Study aim New methodological development/Identification of content (omics approaches)/Technical analysis comparing/optimizing EV-related methods Sample Species Bos taurus Sample Type cheese manufacturing byproducts Separation Method Density gradient Only used for validation of main results Yes Type Discontinuous Number of initial discontinuous layers 5 Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 12 Sample volume (mL) 3 Orientation Top-down Rotor type SW 41 Ti Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 1 Fraction processing None Filtration steps 0.22µm or 0.2µm Other Name other separation method tangential flow filtration EV-subtype Distinction between multiple subtypes Size Used subtypes M.W. >100 kDa Characterization: Protein analysis Protein Concentration Method Fluorometric assay (e.g. Qubit, NanoOrange,...) Western Blot Detected EV-associated proteins TSG101/ HSP70 Not detected EV-associated proteins CD81/ CD63/ CD9/ Alix Detected contaminants Histone H3/ Integrin-beta/ ApoB Proteomics database No Characterization: RNA analysis RNA analysis Type RNA sequencing Database Yes Proteinase treatment No RNAse treatment No Characterization: Lipid analysis Yes Characterization: Particle analysis DLS Report type Mean Reported size (nm) 1108+/-138 NTA Report type Mean Reported size (nm) 102.3+/-6.7 EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 4.12+/-0.525E09 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV210118	2/4	Bos taurus	cheese manufacturing byproducts	DG tangential flow filtration Filtration	Sukreet, Sonal	2021	100%
Study summary Full title Isolation of extracellular vesicles from byproducts of cheesemaking by tangential flow filtration yields heterogeneous fractions of nanoparticles All authors Sonal Sukreet, Camila Pereira Braga, Thuy T. An, Jiri Adamec, Juan Cui, Benjamin Trible, Janos Zempleni Journal Journal of Dairy Science Abstract Extracellular vesicles (EV) in milk, particularly exosomes, have attracted considerable attention as (show more...)Extracellular vesicles (EV) in milk, particularly exosomes, have attracted considerable attention as bioactive food compounds and for their use in drug delivery. The utility of small EV in milk (sMEV) as an animal feed additive and in drug delivery would be enhanced by cost-effective large-scale protocols for the enrichment of sMEV from byproducts in dairy plants. Here, we tested the hypothesis that sMEV may be enriched from byproducts of cheesemaking by tangential flow filtration (EV-FF) and that the sMEV have properties similar to sMEV prepared by ultracentrifugation (sMEV-UC). Three fractions of EV were purified from the whey fraction of cottage cheese making by using EV-FF that passed through a membrane with a 50-kDa cutoff (50 penetrate; 50P), and subfractions of 50P that were retained (100 retentate; 100R) or passed through (100 penetrate; 100P) a membrane with a 100-kDa cutoff; sMEV-UC controls were prepared by serial ultracentrifugation. The abundance of sMEV (<200 nm) was less than 0.3% in EV-FF compared with sMEV-UC (1012/mL of milk). Despite the low EV count, the protein content (mg/mL) of 100R (63 ± 0.02; ± standard deviation) was higher than that of 50P (0.75 ± 0.10), 100P (0.65 ± 0.40), and sMEV-UC (27 ± 0.02). There were 17, 14, 35, and 75 distinct proteins detected by nontargeted mass spectrometry analysis in 50P, 100R, 100P, and sMEV-UC, respectively. Exosome markers CD9, CD63, CD81, HSP-70, PDCD6IP, and TSG101 were detected in control sMEV-UC but not in EV-FF by using targeted mass spectrometry and immunoblot analyses. Negative exosome markers, APOB, β-integrin, and histone H3 were below the limit of detection in EV-FF and control sMEV-UC analyzed by immunoblotting. The abundance of the major milk fat globule protein butyrophilin showed the following pattern: 100R ≫ 100P = 50P > sMEV-UC. More than 100 mature microRNA were detected in sMEV-UC by using sequencing analysis, compared with 36 to 60 microRNA in EV-FF. Only 100R and sMEV-UC yielded mRNA in quantities and qualities sufficient for sequencing analysis; an average of 276,000 and 838,000 reads were mapped to approximately 14,600 and 18,500 genes in 100R and sMEV-UC, respectively. In principal component analysis, microRNA, mRNA, and protein in EV-FF preparations clustered separately from control sMEV-UC. We conclude that under the conditions used here, flow filtration yields a heterogeneous population of milk EV. (hide) EV-METRIC 100% (50th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type cheese manufacturing byproducts Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture DG tangential flow filtration Filtration Protein markers EV: CD81/ Alix/ CD63/ CD9/ HSP70 non-EV: Integrin-beta/ Histone H3/ ApoB Proteomics yes EV density (g/ml) 1.255 Show all info Study aim New methodological development/Identification of content (omics approaches)/Technical analysis comparing/optimizing EV-related methods Sample Species Bos taurus Sample Type cheese manufacturing byproducts Separation Method Density gradient Only used for validation of main results Yes Type Discontinuous Number of initial discontinuous layers 5 Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 12 Sample volume (mL) 3 Orientation Top-down Rotor type SW 41 Ti Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 1 Fraction processing None Filtration steps 0.22µm or 0.2µm Other Name other separation method tangential flow filtration EV-subtype Distinction between multiple subtypes Size Used subtypes M.W. 50 kDa to 100 kDa Characterization: Protein analysis Protein Concentration Method Fluorometric assay (e.g. Qubit, NanoOrange,...) Western Blot Detected EV-associated proteins HSP70 Not detected EV-associated proteins CD81/ CD63/ CD9/ Alix Not detected contaminants Histone H3/ Integrin-beta/ ApoB Proteomics database No Characterization: RNA analysis RNA analysis Type RNAsequencing Database Yes Proteinase treatment No RNAse treatment No Characterization: Lipid analysis Yes Characterization: Particle analysis DLS Report type Mean Reported size (nm) 45+/-17 NTA Report type Mean Reported size (nm) 80.9+/-4.3 EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 2.06+/-0.22E09 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV210118	3/4	Bos taurus	cheese manufacturing byproducts	DG tangential flow filtration Filtration	Sukreet, Sonal	2021	100%
Study summary Full title Isolation of extracellular vesicles from byproducts of cheesemaking by tangential flow filtration yields heterogeneous fractions of nanoparticles All authors Sonal Sukreet, Camila Pereira Braga, Thuy T. An, Jiri Adamec, Juan Cui, Benjamin Trible, Janos Zempleni Journal Journal of Dairy Science Abstract Extracellular vesicles (EV) in milk, particularly exosomes, have attracted considerable attention as (show more...)Extracellular vesicles (EV) in milk, particularly exosomes, have attracted considerable attention as bioactive food compounds and for their use in drug delivery. The utility of small EV in milk (sMEV) as an animal feed additive and in drug delivery would be enhanced by cost-effective large-scale protocols for the enrichment of sMEV from byproducts in dairy plants. Here, we tested the hypothesis that sMEV may be enriched from byproducts of cheesemaking by tangential flow filtration (EV-FF) and that the sMEV have properties similar to sMEV prepared by ultracentrifugation (sMEV-UC). Three fractions of EV were purified from the whey fraction of cottage cheese making by using EV-FF that passed through a membrane with a 50-kDa cutoff (50 penetrate; 50P), and subfractions of 50P that were retained (100 retentate; 100R) or passed through (100 penetrate; 100P) a membrane with a 100-kDa cutoff; sMEV-UC controls were prepared by serial ultracentrifugation. The abundance of sMEV (<200 nm) was less than 0.3% in EV-FF compared with sMEV-UC (1012/mL of milk). Despite the low EV count, the protein content (mg/mL) of 100R (63 ± 0.02; ± standard deviation) was higher than that of 50P (0.75 ± 0.10), 100P (0.65 ± 0.40), and sMEV-UC (27 ± 0.02). There were 17, 14, 35, and 75 distinct proteins detected by nontargeted mass spectrometry analysis in 50P, 100R, 100P, and sMEV-UC, respectively. Exosome markers CD9, CD63, CD81, HSP-70, PDCD6IP, and TSG101 were detected in control sMEV-UC but not in EV-FF by using targeted mass spectrometry and immunoblot analyses. Negative exosome markers, APOB, β-integrin, and histone H3 were below the limit of detection in EV-FF and control sMEV-UC analyzed by immunoblotting. The abundance of the major milk fat globule protein butyrophilin showed the following pattern: 100R ≫ 100P = 50P > sMEV-UC. More than 100 mature microRNA were detected in sMEV-UC by using sequencing analysis, compared with 36 to 60 microRNA in EV-FF. Only 100R and sMEV-UC yielded mRNA in quantities and qualities sufficient for sequencing analysis; an average of 276,000 and 838,000 reads were mapped to approximately 14,600 and 18,500 genes in 100R and sMEV-UC, respectively. In principal component analysis, microRNA, mRNA, and protein in EV-FF preparations clustered separately from control sMEV-UC. We conclude that under the conditions used here, flow filtration yields a heterogeneous population of milk EV. (hide) EV-METRIC 100% (50th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type cheese manufacturing byproducts Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture DG tangential flow filtration Filtration Protein markers EV: CD81/ Alix/ CD63/ CD9/ HSP70 non-EV: Integrin-beta/ Histone H3/ ApoB Proteomics yes EV density (g/ml) 1.255 Show all info Study aim New methodological development/Identification of content (omics approaches)/Technical analysis comparing/optimizing EV-related methods Sample Species Bos taurus Sample Type cheese manufacturing byproducts Separation Method Density gradient Only used for validation of main results Yes Type Discontinuous Number of initial discontinuous layers 5 Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 12 Sample volume (mL) 3 Orientation Top-down Rotor type SW 41 Ti Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 1 Fraction processing None Filtration steps 0.22µm or 0.2µm Other Name other separation method tangential flow filtration EV-subtype Distinction between multiple subtypes Size Used subtypes M.W. >50 kDa Characterization: Protein analysis Protein Concentration Method Fluorometric assay (e.g. Qubit, NanoOrange,...) Western Blot Detected EV-associated proteins HSP70 Not detected EV-associated proteins CD81/ CD63/ CD9/ Alix Not detected contaminants Histone H3/ Integrin-beta/ ApoB Proteomics database No Characterization: RNA analysis RNA analysis Type RNAsequencing Database Yes Proteinase treatment No RNAse treatment No Characterization: Lipid analysis Yes Characterization: Particle analysis DLS Report type Mean Reported size (nm) 157+/-122 NTA Report type Mean Reported size (nm) 90.8 EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 1.44+/-0.144E09 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV210118	4/4	Bos taurus	skim milk	DG (d)(U)C	Sukreet, Sonal	2021	100%
Study summary Full title Isolation of extracellular vesicles from byproducts of cheesemaking by tangential flow filtration yields heterogeneous fractions of nanoparticles All authors Sonal Sukreet, Camila Pereira Braga, Thuy T. An, Jiri Adamec, Juan Cui, Benjamin Trible, Janos Zempleni Journal Journal of Dairy Science Abstract Extracellular vesicles (EV) in milk, particularly exosomes, have attracted considerable attention as (show more...)Extracellular vesicles (EV) in milk, particularly exosomes, have attracted considerable attention as bioactive food compounds and for their use in drug delivery. The utility of small EV in milk (sMEV) as an animal feed additive and in drug delivery would be enhanced by cost-effective large-scale protocols for the enrichment of sMEV from byproducts in dairy plants. Here, we tested the hypothesis that sMEV may be enriched from byproducts of cheesemaking by tangential flow filtration (EV-FF) and that the sMEV have properties similar to sMEV prepared by ultracentrifugation (sMEV-UC). Three fractions of EV were purified from the whey fraction of cottage cheese making by using EV-FF that passed through a membrane with a 50-kDa cutoff (50 penetrate; 50P), and subfractions of 50P that were retained (100 retentate; 100R) or passed through (100 penetrate; 100P) a membrane with a 100-kDa cutoff; sMEV-UC controls were prepared by serial ultracentrifugation. The abundance of sMEV (<200 nm) was less than 0.3% in EV-FF compared with sMEV-UC (1012/mL of milk). Despite the low EV count, the protein content (mg/mL) of 100R (63 ± 0.02; ± standard deviation) was higher than that of 50P (0.75 ± 0.10), 100P (0.65 ± 0.40), and sMEV-UC (27 ± 0.02). There were 17, 14, 35, and 75 distinct proteins detected by nontargeted mass spectrometry analysis in 50P, 100R, 100P, and sMEV-UC, respectively. Exosome markers CD9, CD63, CD81, HSP-70, PDCD6IP, and TSG101 were detected in control sMEV-UC but not in EV-FF by using targeted mass spectrometry and immunoblot analyses. Negative exosome markers, APOB, β-integrin, and histone H3 were below the limit of detection in EV-FF and control sMEV-UC analyzed by immunoblotting. The abundance of the major milk fat globule protein butyrophilin showed the following pattern: 100R ≫ 100P = 50P > sMEV-UC. More than 100 mature microRNA were detected in sMEV-UC by using sequencing analysis, compared with 36 to 60 microRNA in EV-FF. Only 100R and sMEV-UC yielded mRNA in quantities and qualities sufficient for sequencing analysis; an average of 276,000 and 838,000 reads were mapped to approximately 14,600 and 18,500 genes in 100R and sMEV-UC, respectively. In principal component analysis, microRNA, mRNA, and protein in EV-FF preparations clustered separately from control sMEV-UC. We conclude that under the conditions used here, flow filtration yields a heterogeneous population of milk EV. (hide) EV-METRIC 100% (50th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type skim milk Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture DG (d)(U)C Protein markers EV: TSG101/ CD63/ CD81/ Alix/ HSP70/ CD9 non-EV: Integrin-beta/ Histone H3/ ApoB Proteomics yes EV density (g/ml) 1.255 Show all info Study aim New methodological development/Identification of content (omics approaches)/Technical analysis comparing/optimizing EV-related methods Sample Species Bos taurus Sample Type skim milk Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Between 50,000 g and 100,000 g Pelleting performed Yes Pelleting: time(min) 90 Pelleting: rotor type F37L-8x100 Pelleting: speed (g) 120000 Wash: volume per pellet (ml) 1 Wash: time (min) 90 Wash: Rotor Type F37L-8x100 Wash: speed (g) 120000 Density gradient Only used for validation of main results Yes Type Discontinuous Number of initial discontinuous layers 5 Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 12 Sample volume (mL) 3 Orientation Top-down Rotor type SW 41 Ti Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 1 Fraction processing None Characterization: Protein analysis Protein Concentration Method Fluorometric assay (e.g. Qubit, NanoOrange,...) Western Blot Detected EV-associated proteins Alix/ CD9/ CD63/ TSG101/ HSP70/ CD81 Not detected contaminants Histone H3/ Integrin-beta/ ApoB Proteomics database No Characterization: RNA analysis RNA analysis Type RNAsequencing Database Yes Proteinase treatment No RNAse treatment No Characterization: Lipid analysis Yes Characterization: Particle analysis DLS Report type Mean Reported size (nm) 115+/-31 NTA Report type Mean Reported size (nm) 106.6 EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 1.42+/-0.0536E14 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV210043	1/2	Homo sapiens	hcMEC/D3	DG (d)(U)C UF Filtration	Fikatas, Antonios	2021	100%
Study summary Full title Deciphering the Role of Extracellular Vesicles Derived from ZIKV-Infected hcMEC/D3 Cells on the Blood-Brain Barrier System All authors Antonios Fikatas, Jonas Dehairs, Sam Noppen, Jordi Doijen, Frank Vanderhoydonc, Eef Meyen, Johannes V Swinnen, Christophe Pannecouque, Dominique Schols Journal Viruses Abstract To date, no vaccines or antivirals are available against Zika virus (ZIKV). In addition, the mechani (show more...)To date, no vaccines or antivirals are available against Zika virus (ZIKV). In addition, the mechanisms underlying ZIKV-associated pathogenesis of the central nervous system (CNS) are largely unexplored. Getting more insight into the cellular pathways that ZIKV recruits to facilitate infection of susceptible cells will be crucial for establishing an effective treatment strategy. In general, cells secrete a number of vesicles, known as extracellular vesicles (EVs), in response to viral infections. These EVs serve as intercellular communicators. Here, we investigated the role of EVs derived from ZIKV-infected human brain microvascular endothelial cells on the blood–brain barrier (BBB) system. We demonstrated that ZIKV-infected EVs (IEVs) can incorporate viral components, including ZIKV RNA, NS1, and E-protein, and further transfer them to several types of CNS cells. Using label-free impedance-based biosensing, we observed that ZIKV and IEVs can temporally disturb the monolayer integrity of BBB-mimicking cells, possibly by inducing structural rearrangements of the adherent protein VE-cadherin (immunofluorescence staining). Finally, differences in the lipidomic profile between EVs and their parental cells possibly suggest a preferential sorting mechanism of specific lipid species into the vesicles. To conclude, these data suggest that IEVs could be postulated as vehicles (Trojan horse) for ZIKV transmission via the BBB. (hide) EV-METRIC 100% (99th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture DG (d)(U)C UF Filtration Protein markers EV: TSG101/ Alix/ CD63 non-EV: Calnexin Proteomics no EV density (g/ml) 1.06-1.16 Show all info Study aim Function/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells hcMEC/D3 EV-harvesting Medium EV-depleted medium Preparation of EDS Commercial EDS Cell viability (%) NA Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Pelleting performed Yes Pelleting: time(min) 1260 Pelleting: rotor type TH-641 Pelleting: speed (g) 100 000 Wash: volume per pellet (ml) 10 Wash: time (min) 180 Wash: Rotor Type TH-641 Wash: speed (g) 100 000 Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 10 Sample volume (mL) 1 Orientation Top-down Rotor type TH-641 Speed (g) 100 000 Duration (min) 1080 Fraction volume (mL) 1,5 Fraction processing None Filtration steps 0.22µm or 0.2µm Ultra filtration Cut-off size (kDa) 10 Membrane type Regenerated cellulose Characterization: Protein analysis Protein Concentration Method Bradford Western Blot Detected EV-associated proteins CD63/ TSG101/ Alix Not detected contaminants Calnexin Characterization: Lipid analysis Yes Characterization: Particle analysis NTA Report type Mean Reported size (nm) 165 EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 1,50E+08 EM EM-type Transmission-EM Image type Close-up, Wide-field Report size (nm) 130

	EV200102	1/7	Homo sapiens	THP1	DG (d)(U)C qEV	Tóth, Eszter	2021	100%
Study summary Full title Formation of a protein corona on the surface of extracellular vesicles in blood plasma All authors Eszter Á Tóth, Lilla Turiák, Tamás Visnovitz, Csaba Cserép, Anett Mázló, Barbara W Sódar, András I Försönits, Gábor Petővári, Anna Sebestyén, Zsolt Komlósi, László Drahos, Ágnes Kittel, György Nagy, Attila Bácsi, Ádám Dénes, Yong Song Gho, Katalin É Szabó-Taylor, Edit I Buzás Journal J Extracell Vesicles Abstract In this study we tested whether a protein corona is formed around extracellular vesicles (EVs) in bl (show more...)In this study we tested whether a protein corona is formed around extracellular vesicles (EVs) in blood plasma. We isolated medium-sized nascent EVs of THP1 cells as well as of Optiprep-purified platelets, and incubated them in EV-depleted blood plasma from healthy subjects and from patients with rheumatoid arthritis. EVs were subjected to differential centrifugation, size exclusion chromatography, or density gradient ultracentrifugation followed by mass spectrometry. Plasma protein-coated EVs had a higher density compared to the nascent ones and carried numerous newly associated proteins. Interactions between plasma proteins and EVs were confirmed by confocal microscopy, capillary Western immunoassay, immune electron microscopy and flow cytometry. We identified nine shared EV corona proteins (ApoA1, ApoB, ApoC3, ApoE, complement factors 3 and 4B, fibrinogen α-chain, immunoglobulin heavy constant γ2 and γ4 chains), which appear to be common corona proteins among EVs, viruses and artificial nanoparticles in blood plasma. An unexpected finding of this study was the high overlap of the composition of the protein corona with blood plasma protein aggregates. This is explained by our finding that besides a diffuse, patchy protein corona, large protein aggregates also associate with the surface of EVs. However, while EVs with an external plasma protein cargo induced an increased expression of TNF-α, IL-6, CD83, CD86 and HLA-DR of human monocyte-derived dendritic cells, EV-free protein aggregates had no effect. In conclusion, our data may shed new light on the origin of the commonly reported plasma protein 'contamination' of EV preparations and may add a new perspective to EV research. (hide) EV-METRIC 100% (99th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture Density gradient (Differential) (ultra)centrifugation Commercial method Protein markers EV: CD63/ Phosphatydilserine non-EV: None Proteomics yes EV density (g/ml) 1.10-1.15 Show all info Study aim Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells THP1 EV-harvesting Medium Serum free medium Cell viability (%) 93 Cell count 80000000 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 10,000 g and 50,000 g Pelleting performed Yes Pelleting: time(min) 40 Pelleting: rotor type FA-45-24-11 Pelleting: speed (g) 12500 Wash: volume per pellet (ml) 1 Wash: time (min) 40 Wash: Rotor Type FA-45-24-11 Wash: speed (g) 12500 Density gradient Only used for validation of main results Yes Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 4.5 Sample volume (mL) 0.5 Orientation Top-down Rotor type MLS-50 Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 0.5 Fraction processing Centrifugation Pelleting: volume per fraction 2 Pelleting: duration (min) 80 Pelleting: rotor type FA-45-24-11 Pelleting: speed (g) 12500 Commercial kit qEV Characterization: Protein analysis Protein Concentration Method microBCA Flow cytometry Type of Flow cytometry FACS Calibur Calibration bead size The vesicular gate was set using Megamix Beads (Bi Detected EV-associated proteins Phosphatydilserine Proteomics database Yes: Detected EV-associated proteins CD63 Characterization: Lipid analysis Yes Characterization: Particle analysis TRPS Report type Modus Reported size (nm) 244 Particle analysis: flow cytometry Flow cytometer type FACS Calibur Hardware adjustment Calibration bead size 0.160;0.200;0.240;0.500 Report type Not Reported EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV200099	1/8	Bos taurus	milk	(d)(U)C DG	Kleinjan, Marije	2021	100%
Study summary Full title Regular Industrial Processing of Bovine Milk Impacts the Integrity and Molecular Composition of Extracellular Vesicles All authors Marije Kleinjan, Martijn Jc van Herwijnen, Sten Fwm Libregts, Rj Joost van Neerven, Anouk L Feitsma, Marca Hm Wauben Journal J Nutr Abstract Background: Bovine milk contains extracellular vesicles (EVs), which act as mediators of intercellul (show more...)Background: Bovine milk contains extracellular vesicles (EVs), which act as mediators of intercellular communication by regulating the recipients’ cellular processes via their selectively incorporated bioactive molecules. Because some of these EV components are evolutionarily conserved, EVs present in commercial milk might have the potential to regulate cellular processes in human consumers. Objectives: Because commercial milk is subjected to industrial processing, we investigated its effect on the number and integrity of isolated milk EVs and their bioactive components. For this, we compared EVs isolated from raw bovine milk with EVs isolated from different types of commercial milk, including pasteurized milk, either homogenized or not, and ultra heat treated (UHT) milk. Methods: EVs were separated from other milk components by differential centrifugation, followed by density gradient ultracentrifugation. EVs from different milk types were compared by single-particle high-resolution fluorescence-based flow cytometry to determine EV numbers, Cryo-electron microscopy to visualize EV integrity and morphology, western blot analysis to investigate EV-associated protein cargo, and RNA analysis to assess total small RNA concentration and milk-EV-specific microRNA expression. Results: In UHT milk, we could not detect intact EVs. Interestingly, although pasteurization (irrespective of homogenization) did not affect mean ± SD EV numbers (3.4 × 108 ± 1.2 × 108–2.8 × 108 ± 0.3 × 107 compared with 3.1 × 108 ± 1.2 × 108 in raw milk), it affected EV integrity and appearance, altered their protein signature, and resulted in a loss of milk-EV-associated RNAs (from 40.2 ± 3.4 ng/μL in raw milk to 17.7 ± 5.4–23.3 ± 10.0 mg/μL in processed milk, P < 0.05). Conclusions: Commercial milk, that has been heated by either pasteurization or UHT, contains fewer or no intact EVs, respectively. Although most EVs seemed resistant to pasteurization based on particle numbers, their integrity was affected and their molecular composition was altered. Thus, the possible transfer of bioactive components via bovine milk EVs to human consumers is likely diminished or altered in heat-treated commercial milk. (hide) EV-METRIC 100% (93rd percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type milk Sample origin raw Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C DG Protein markers EV: MFG-E8/ CD63/ TSG101/ Flotillin1/ CD9 non-EV: beta-lactoglobulin/ beta-casein Proteomics no EV density (g/ml) 1.20-1.24 Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Bos taurus Sample Type milk Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Pelleting performed No Density gradient Type Continuous Lowest density fraction 0.4M Highest density fraction 2.5M Total gradient volume, incl. sample (mL) 12 Sample volume (mL) 6.5 Orientation Top-down Rotor type SW 40 Ti Speed (g) 192000 Duration (min) 1080 Fraction volume (mL) 0.5 Fraction processing Centrifugation Pelleting: volume per fraction 1.5 Pelleting: duration (min) 65 Pelleting: rotor type SW 40 Ti Pelleting: speed (g) 100000 EV-subtype Distinction between multiple subtypes Density Used subtypes 1.20-1.24 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Detected EV-associated proteins Flotillin1/ CD9/ CD63/ MFG-E8/ TSG101 Not detected contaminants beta-casein/ beta-lactoglobulin Characterization: RNA analysis RNA analysis Type (RT)(q)PCR Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis Particle analysis: flow cytometry Flow cytometer type BD Influx cell sorter (BD Biosciences, San Jose, CA, USA) Hardware adjustment High-resolution flow cytometric analysis of PKH67-stained samples was performed on a BD Influx cell sorter (BD Biosciences, San Jose, CA, USA) that was dedicated and optimized for detection of submicron-sized particles Calibration bead size 0.1 EV concentration Yes EM EM-type Cryo-EM Image type Close-up, Wide-field Report size (nm) 200

	EV200099	2/8	Bos taurus	milk	(d)(U)C DG	Kleinjan, Marije	2021	100%
Study summary Full title Regular Industrial Processing of Bovine Milk Impacts the Integrity and Molecular Composition of Extracellular Vesicles All authors Marije Kleinjan, Martijn Jc van Herwijnen, Sten Fwm Libregts, Rj Joost van Neerven, Anouk L Feitsma, Marca Hm Wauben Journal J Nutr Abstract Background: Bovine milk contains extracellular vesicles (EVs), which act as mediators of intercellul (show more...)Background: Bovine milk contains extracellular vesicles (EVs), which act as mediators of intercellular communication by regulating the recipients’ cellular processes via their selectively incorporated bioactive molecules. Because some of these EV components are evolutionarily conserved, EVs present in commercial milk might have the potential to regulate cellular processes in human consumers. Objectives: Because commercial milk is subjected to industrial processing, we investigated its effect on the number and integrity of isolated milk EVs and their bioactive components. For this, we compared EVs isolated from raw bovine milk with EVs isolated from different types of commercial milk, including pasteurized milk, either homogenized or not, and ultra heat treated (UHT) milk. Methods: EVs were separated from other milk components by differential centrifugation, followed by density gradient ultracentrifugation. EVs from different milk types were compared by single-particle high-resolution fluorescence-based flow cytometry to determine EV numbers, Cryo-electron microscopy to visualize EV integrity and morphology, western blot analysis to investigate EV-associated protein cargo, and RNA analysis to assess total small RNA concentration and milk-EV-specific microRNA expression. Results: In UHT milk, we could not detect intact EVs. Interestingly, although pasteurization (irrespective of homogenization) did not affect mean ± SD EV numbers (3.4 × 108 ± 1.2 × 108–2.8 × 108 ± 0.3 × 107 compared with 3.1 × 108 ± 1.2 × 108 in raw milk), it affected EV integrity and appearance, altered their protein signature, and resulted in a loss of milk-EV-associated RNAs (from 40.2 ± 3.4 ng/μL in raw milk to 17.7 ± 5.4–23.3 ± 10.0 mg/μL in processed milk, P < 0.05). Conclusions: Commercial milk, that has been heated by either pasteurization or UHT, contains fewer or no intact EVs, respectively. Although most EVs seemed resistant to pasteurization based on particle numbers, their integrity was affected and their molecular composition was altered. Thus, the possible transfer of bioactive components via bovine milk EVs to human consumers is likely diminished or altered in heat-treated commercial milk. (hide) EV-METRIC 100% (93rd percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type milk Sample origin raw Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C DG Protein markers EV: MFG-E8/ CD63/ TSG101/ Flotillin1/ CD9 non-EV: beta-lactoglobulin/ beta-casein Proteomics no EV density (g/ml) 1.18-1.13 Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Bos taurus Sample Type milk Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Pelleting performed No Density gradient Type Continuous Lowest density fraction 0.4M Highest density fraction 2.5M Total gradient volume, incl. sample (mL) 12 Sample volume (mL) 6.5 Orientation Top-down Rotor type SW 40 Ti Speed (g) 192000 Duration (min) 1080 Fraction volume (mL) 0.5 Fraction processing Centrifugation Pelleting: volume per fraction 1.5 Pelleting: duration (min) 65 Pelleting: rotor type SW 40 Ti Pelleting: speed (g) 100000 EV-subtype Distinction between multiple subtypes Density Used subtypes 1.18-1.13 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Detected EV-associated proteins Flotillin1/ CD9/ CD63/ MFG-E8/ TSG101 Not detected contaminants beta-casein/ beta-lactoglobulin Characterization: RNA analysis RNA analysis Type (RT)(q)PCR Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis Particle analysis: flow cytometry Flow cytometer type BD Influx cell sorter (BD Biosciences, San Jose, CA, USA) Hardware adjustment High-resolution flow cytometric analysis of PKH67-stained samples was performed on a BD Influx cell sorter (BD Biosciences, San Jose, CA, USA) that was dedicated and optimized for detection of submicron-sized particles Calibration bead size 0.1 Report type Not Reported EV concentration Yes EM EM-type Cryo-EM Image type Close-up, Wide-field Report size (nm) 200

	EV200099	3/8	Bos taurus	milk	(d)(U)C DG	Kleinjan, Marije	2021	100%
Study summary Full title Regular Industrial Processing of Bovine Milk Impacts the Integrity and Molecular Composition of Extracellular Vesicles All authors Marije Kleinjan, Martijn Jc van Herwijnen, Sten Fwm Libregts, Rj Joost van Neerven, Anouk L Feitsma, Marca Hm Wauben Journal J Nutr Abstract Background: Bovine milk contains extracellular vesicles (EVs), which act as mediators of intercellul (show more...)Background: Bovine milk contains extracellular vesicles (EVs), which act as mediators of intercellular communication by regulating the recipients’ cellular processes via their selectively incorporated bioactive molecules. Because some of these EV components are evolutionarily conserved, EVs present in commercial milk might have the potential to regulate cellular processes in human consumers. Objectives: Because commercial milk is subjected to industrial processing, we investigated its effect on the number and integrity of isolated milk EVs and their bioactive components. For this, we compared EVs isolated from raw bovine milk with EVs isolated from different types of commercial milk, including pasteurized milk, either homogenized or not, and ultra heat treated (UHT) milk. Methods: EVs were separated from other milk components by differential centrifugation, followed by density gradient ultracentrifugation. EVs from different milk types were compared by single-particle high-resolution fluorescence-based flow cytometry to determine EV numbers, Cryo-electron microscopy to visualize EV integrity and morphology, western blot analysis to investigate EV-associated protein cargo, and RNA analysis to assess total small RNA concentration and milk-EV-specific microRNA expression. Results: In UHT milk, we could not detect intact EVs. Interestingly, although pasteurization (irrespective of homogenization) did not affect mean ± SD EV numbers (3.4 × 108 ± 1.2 × 108–2.8 × 108 ± 0.3 × 107 compared with 3.1 × 108 ± 1.2 × 108 in raw milk), it affected EV integrity and appearance, altered their protein signature, and resulted in a loss of milk-EV-associated RNAs (from 40.2 ± 3.4 ng/μL in raw milk to 17.7 ± 5.4–23.3 ± 10.0 mg/μL in processed milk, P < 0.05). Conclusions: Commercial milk, that has been heated by either pasteurization or UHT, contains fewer or no intact EVs, respectively. Although most EVs seemed resistant to pasteurization based on particle numbers, their integrity was affected and their molecular composition was altered. Thus, the possible transfer of bioactive components via bovine milk EVs to human consumers is likely diminished or altered in heat-treated commercial milk. (hide) EV-METRIC 100% (93rd percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type milk Sample origin pasteurized Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C DG Protein markers EV: MFG-E8/ CD63/ TSG101/ Flotillin1/ CD9 non-EV: beta-lactoglobulin/ beta-casein Proteomics no EV density (g/ml) 1.20-1.24 Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Bos taurus Sample Type milk Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Pelleting performed No Density gradient Type Continuous Lowest density fraction 0.4M Highest density fraction 2.5M Total gradient volume, incl. sample (mL) 12 Sample volume (mL) 6.5 Orientation Top-down Rotor type SW 40 Ti Speed (g) 192000 Duration (min) 1080 Fraction volume (mL) 0.5 Fraction processing Centrifugation Pelleting: volume per fraction 1.5 Pelleting: duration (min) 65 Pelleting: rotor type SW 40 Ti Pelleting: speed (g) 100000 EV-subtype Distinction between multiple subtypes Density Used subtypes 1.20-1.24 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Detected EV-associated proteins Flotillin1/ CD9/ CD63/ MFG-E8/ TSG101 Not detected contaminants beta-casein/ beta-lactoglobulin Characterization: RNA analysis RNA analysis Type (RT)(q)PCR Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis Particle analysis: flow cytometry Flow cytometer type BD Influx cell sorter (BD Biosciences, San Jose, CA, USA) Hardware adjustment High-resolution flow cytometric analysis of PKH67-stained samples was performed on a BD Influx cell sorter (BD Biosciences, San Jose, CA, USA) that was dedicated and optimized for detection of submicron-sized particles Calibration bead size 0.1 EV concentration Yes EM EM-type Cryo-EM Image type Close-up, Wide-field Report size (nm) 200

	EV200099	4/8	Bos taurus	milk	(d)(U)C DG	Kleinjan, Marije	2021	100%
Study summary Full title Regular Industrial Processing of Bovine Milk Impacts the Integrity and Molecular Composition of Extracellular Vesicles All authors Marije Kleinjan, Martijn Jc van Herwijnen, Sten Fwm Libregts, Rj Joost van Neerven, Anouk L Feitsma, Marca Hm Wauben Journal J Nutr Abstract Background: Bovine milk contains extracellular vesicles (EVs), which act as mediators of intercellul (show more...)Background: Bovine milk contains extracellular vesicles (EVs), which act as mediators of intercellular communication by regulating the recipients’ cellular processes via their selectively incorporated bioactive molecules. Because some of these EV components are evolutionarily conserved, EVs present in commercial milk might have the potential to regulate cellular processes in human consumers. Objectives: Because commercial milk is subjected to industrial processing, we investigated its effect on the number and integrity of isolated milk EVs and their bioactive components. For this, we compared EVs isolated from raw bovine milk with EVs isolated from different types of commercial milk, including pasteurized milk, either homogenized or not, and ultra heat treated (UHT) milk. Methods: EVs were separated from other milk components by differential centrifugation, followed by density gradient ultracentrifugation. EVs from different milk types were compared by single-particle high-resolution fluorescence-based flow cytometry to determine EV numbers, Cryo-electron microscopy to visualize EV integrity and morphology, western blot analysis to investigate EV-associated protein cargo, and RNA analysis to assess total small RNA concentration and milk-EV-specific microRNA expression. Results: In UHT milk, we could not detect intact EVs. Interestingly, although pasteurization (irrespective of homogenization) did not affect mean ± SD EV numbers (3.4 × 108 ± 1.2 × 108–2.8 × 108 ± 0.3 × 107 compared with 3.1 × 108 ± 1.2 × 108 in raw milk), it affected EV integrity and appearance, altered their protein signature, and resulted in a loss of milk-EV-associated RNAs (from 40.2 ± 3.4 ng/μL in raw milk to 17.7 ± 5.4–23.3 ± 10.0 mg/μL in processed milk, P < 0.05). Conclusions: Commercial milk, that has been heated by either pasteurization or UHT, contains fewer or no intact EVs, respectively. Although most EVs seemed resistant to pasteurization based on particle numbers, their integrity was affected and their molecular composition was altered. Thus, the possible transfer of bioactive components via bovine milk EVs to human consumers is likely diminished or altered in heat-treated commercial milk. (hide) EV-METRIC 100% (93rd percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type milk Sample origin pasteurized Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C DG Protein markers EV: MFG-E8/ CD63/ TSG101/ Flotillin1/ CD9 non-EV: beta-lactoglobulin/ beta-casein Proteomics no EV density (g/ml) 1.18-1.13 Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Bos taurus Sample Type milk Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Pelleting performed No Density gradient Type Continuous Lowest density fraction 0.4M Highest density fraction 2.5M Total gradient volume, incl. sample (mL) 12 Sample volume (mL) 6.5 Orientation Top-down Rotor type SW 40 Ti Speed (g) 192000 Duration (min) 1080 Fraction volume (mL) 0.5 Fraction processing Centrifugation Pelleting: volume per fraction 1.5 Pelleting: duration (min) 65 Pelleting: rotor type SW 40 Ti Pelleting: speed (g) 100000 EV-subtype Distinction between multiple subtypes Density Used subtypes 1.18-1.13 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Detected EV-associated proteins Flotillin1/ CD9/ CD63/ MFG-E8/ TSG101 Not detected contaminants beta-casein/ beta-lactoglobulin Characterization: RNA analysis RNA analysis Type (RT)(q)PCR Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis Particle analysis: flow cytometry Flow cytometer type BD Influx cell sorter (BD Biosciences, San Jose, CA, USA) Hardware adjustment High-resolution flow cytometric analysis of PKH67-stained samples was performed on a BD Influx cell sorter (BD Biosciences, San Jose, CA, USA) that was dedicated and optimized for detection of submicron-sized particles Calibration bead size 0.1 Report type Not Reported EV concentration Yes EM EM-type Cryo-EM Image type Close-up, Wide-field Report size (nm) 200

	EV200099	5/8	Bos taurus	milk	(d)(U)C DG	Kleinjan, Marije	2021	100%
Study summary Full title Regular Industrial Processing of Bovine Milk Impacts the Integrity and Molecular Composition of Extracellular Vesicles All authors Marije Kleinjan, Martijn Jc van Herwijnen, Sten Fwm Libregts, Rj Joost van Neerven, Anouk L Feitsma, Marca Hm Wauben Journal J Nutr Abstract Background: Bovine milk contains extracellular vesicles (EVs), which act as mediators of intercellul (show more...)Background: Bovine milk contains extracellular vesicles (EVs), which act as mediators of intercellular communication by regulating the recipients’ cellular processes via their selectively incorporated bioactive molecules. Because some of these EV components are evolutionarily conserved, EVs present in commercial milk might have the potential to regulate cellular processes in human consumers. Objectives: Because commercial milk is subjected to industrial processing, we investigated its effect on the number and integrity of isolated milk EVs and their bioactive components. For this, we compared EVs isolated from raw bovine milk with EVs isolated from different types of commercial milk, including pasteurized milk, either homogenized or not, and ultra heat treated (UHT) milk. Methods: EVs were separated from other milk components by differential centrifugation, followed by density gradient ultracentrifugation. EVs from different milk types were compared by single-particle high-resolution fluorescence-based flow cytometry to determine EV numbers, Cryo-electron microscopy to visualize EV integrity and morphology, western blot analysis to investigate EV-associated protein cargo, and RNA analysis to assess total small RNA concentration and milk-EV-specific microRNA expression. Results: In UHT milk, we could not detect intact EVs. Interestingly, although pasteurization (irrespective of homogenization) did not affect mean ± SD EV numbers (3.4 × 108 ± 1.2 × 108–2.8 × 108 ± 0.3 × 107 compared with 3.1 × 108 ± 1.2 × 108 in raw milk), it affected EV integrity and appearance, altered their protein signature, and resulted in a loss of milk-EV-associated RNAs (from 40.2 ± 3.4 ng/μL in raw milk to 17.7 ± 5.4–23.3 ± 10.0 mg/μL in processed milk, P < 0.05). Conclusions: Commercial milk, that has been heated by either pasteurization or UHT, contains fewer or no intact EVs, respectively. Although most EVs seemed resistant to pasteurization based on particle numbers, their integrity was affected and their molecular composition was altered. Thus, the possible transfer of bioactive components via bovine milk EVs to human consumers is likely diminished or altered in heat-treated commercial milk. (hide) EV-METRIC 100% (93rd percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type milk Sample origin pasteurized and homogenized Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C DG Protein markers EV: MFG-E8/ CD63/ TSG101/ Flotillin1/ CD9 non-EV: beta-lactoglobulin/ beta-casein Proteomics no EV density (g/ml) 1.20-1.24 Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Bos taurus Sample Type milk Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Pelleting performed No Density gradient Type Continuous Lowest density fraction 0.4M Highest density fraction 2.5M Total gradient volume, incl. sample (mL) 12 Sample volume (mL) 6.5 Orientation Top-down Rotor type SW 40 Ti Speed (g) 192000 Duration (min) 1080 Fraction volume (mL) 0.5 Fraction processing Centrifugation Pelleting: volume per fraction 1.5 Pelleting: duration (min) 65 Pelleting: rotor type SW 40 Ti Pelleting: speed (g) 100000 EV-subtype Distinction between multiple subtypes Density Used subtypes 1.20-1.24 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Detected EV-associated proteins Flotillin1/ CD9/ CD63/ MFG-E8/ TSG101 Not detected contaminants beta-casein/ beta-lactoglobulin Characterization: RNA analysis RNA analysis Type (RT)(q)PCR Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis Particle analysis: flow cytometry Flow cytometer type BD Influx cell sorter (BD Biosciences, San Jose, CA, USA) Hardware adjustment High-resolution flow cytometric analysis of PKH67-stained samples was performed on a BD Influx cell sorter (BD Biosciences, San Jose, CA, USA) that was dedicated and optimized for detection of submicron-sized particles Calibration bead size 0.1 EV concentration Yes EM EM-type Cryo-EM Image type Close-up, Wide-field Report size (nm) 200

	EV200099	6/8	Bos taurus	milk	(d)(U)C DG	Kleinjan, Marije	2021	100%
Study summary Full title Regular Industrial Processing of Bovine Milk Impacts the Integrity and Molecular Composition of Extracellular Vesicles All authors Marije Kleinjan, Martijn Jc van Herwijnen, Sten Fwm Libregts, Rj Joost van Neerven, Anouk L Feitsma, Marca Hm Wauben Journal J Nutr Abstract Background: Bovine milk contains extracellular vesicles (EVs), which act as mediators of intercellul (show more...)Background: Bovine milk contains extracellular vesicles (EVs), which act as mediators of intercellular communication by regulating the recipients’ cellular processes via their selectively incorporated bioactive molecules. Because some of these EV components are evolutionarily conserved, EVs present in commercial milk might have the potential to regulate cellular processes in human consumers. Objectives: Because commercial milk is subjected to industrial processing, we investigated its effect on the number and integrity of isolated milk EVs and their bioactive components. For this, we compared EVs isolated from raw bovine milk with EVs isolated from different types of commercial milk, including pasteurized milk, either homogenized or not, and ultra heat treated (UHT) milk. Methods: EVs were separated from other milk components by differential centrifugation, followed by density gradient ultracentrifugation. EVs from different milk types were compared by single-particle high-resolution fluorescence-based flow cytometry to determine EV numbers, Cryo-electron microscopy to visualize EV integrity and morphology, western blot analysis to investigate EV-associated protein cargo, and RNA analysis to assess total small RNA concentration and milk-EV-specific microRNA expression. Results: In UHT milk, we could not detect intact EVs. Interestingly, although pasteurization (irrespective of homogenization) did not affect mean ± SD EV numbers (3.4 × 108 ± 1.2 × 108–2.8 × 108 ± 0.3 × 107 compared with 3.1 × 108 ± 1.2 × 108 in raw milk), it affected EV integrity and appearance, altered their protein signature, and resulted in a loss of milk-EV-associated RNAs (from 40.2 ± 3.4 ng/μL in raw milk to 17.7 ± 5.4–23.3 ± 10.0 mg/μL in processed milk, P < 0.05). Conclusions: Commercial milk, that has been heated by either pasteurization or UHT, contains fewer or no intact EVs, respectively. Although most EVs seemed resistant to pasteurization based on particle numbers, their integrity was affected and their molecular composition was altered. Thus, the possible transfer of bioactive components via bovine milk EVs to human consumers is likely diminished or altered in heat-treated commercial milk. (hide) EV-METRIC 100% (93rd percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type milk Sample origin pasteurized and homogenized Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C DG Protein markers EV: MFG-E8/ CD63/ TSG101/ Flotillin1/ CD9 non-EV: beta-lactoglobulin/ beta-casein Proteomics no EV density (g/ml) 1.18-1.13 Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Bos taurus Sample Type milk Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Pelleting performed No Density gradient Type Continuous Lowest density fraction 0.4M Highest density fraction 2.5M Total gradient volume, incl. sample (mL) 12 Sample volume (mL) 6.5 Orientation Top-down Rotor type SW 40 Ti Speed (g) 192000 Duration (min) 1080 Fraction volume (mL) 0.5 Fraction processing Centrifugation Pelleting: volume per fraction 1.5 Pelleting: duration (min) 65 Pelleting: rotor type SW 40 Ti Pelleting: speed (g) 100000 EV-subtype Distinction between multiple subtypes Density Used subtypes 1.18-1.13 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Detected EV-associated proteins Flotillin1/ CD9/ CD63/ MFG-E8/ TSG101 Not detected contaminants beta-casein/ beta-lactoglobulin Characterization: RNA analysis RNA analysis Type (RT)(q)PCR Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis Particle analysis: flow cytometry Flow cytometer type BD Influx cell sorter (BD Biosciences, San Jose, CA, USA) Hardware adjustment High-resolution flow cytometric analysis of PKH67-stained samples was performed on a BD Influx cell sorter (BD Biosciences, San Jose, CA, USA) that was dedicated and optimized for detection of submicron-sized particles Calibration bead size 0.1 Report type Not Reported EV concentration Yes EM EM-type Cryo-EM Image type Close-up, Wide-field Report size (nm) 200

	EV200099	7/8	Bos taurus	milk	(d)(U)C DG	Kleinjan, Marije	2021	100%
Study summary Full title Regular Industrial Processing of Bovine Milk Impacts the Integrity and Molecular Composition of Extracellular Vesicles All authors Marije Kleinjan, Martijn Jc van Herwijnen, Sten Fwm Libregts, Rj Joost van Neerven, Anouk L Feitsma, Marca Hm Wauben Journal J Nutr Abstract Background: Bovine milk contains extracellular vesicles (EVs), which act as mediators of intercellul (show more...)Background: Bovine milk contains extracellular vesicles (EVs), which act as mediators of intercellular communication by regulating the recipients’ cellular processes via their selectively incorporated bioactive molecules. Because some of these EV components are evolutionarily conserved, EVs present in commercial milk might have the potential to regulate cellular processes in human consumers. Objectives: Because commercial milk is subjected to industrial processing, we investigated its effect on the number and integrity of isolated milk EVs and their bioactive components. For this, we compared EVs isolated from raw bovine milk with EVs isolated from different types of commercial milk, including pasteurized milk, either homogenized or not, and ultra heat treated (UHT) milk. Methods: EVs were separated from other milk components by differential centrifugation, followed by density gradient ultracentrifugation. EVs from different milk types were compared by single-particle high-resolution fluorescence-based flow cytometry to determine EV numbers, Cryo-electron microscopy to visualize EV integrity and morphology, western blot analysis to investigate EV-associated protein cargo, and RNA analysis to assess total small RNA concentration and milk-EV-specific microRNA expression. Results: In UHT milk, we could not detect intact EVs. Interestingly, although pasteurization (irrespective of homogenization) did not affect mean ± SD EV numbers (3.4 × 108 ± 1.2 × 108–2.8 × 108 ± 0.3 × 107 compared with 3.1 × 108 ± 1.2 × 108 in raw milk), it affected EV integrity and appearance, altered their protein signature, and resulted in a loss of milk-EV-associated RNAs (from 40.2 ± 3.4 ng/μL in raw milk to 17.7 ± 5.4–23.3 ± 10.0 mg/μL in processed milk, P < 0.05). Conclusions: Commercial milk, that has been heated by either pasteurization or UHT, contains fewer or no intact EVs, respectively. Although most EVs seemed resistant to pasteurization based on particle numbers, their integrity was affected and their molecular composition was altered. Thus, the possible transfer of bioactive components via bovine milk EVs to human consumers is likely diminished or altered in heat-treated commercial milk. (hide) EV-METRIC 100% (93rd percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type milk Sample origin UHT Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C DG Protein markers EV: MFG-E8/ CD63/ TSG101/ Flotillin1/ CD9 non-EV: beta-lactoglobulin/ beta-casein Proteomics no EV density (g/ml) 1.20-1.24 Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Bos taurus Sample Type milk Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Pelleting performed No Density gradient Type Continuous Lowest density fraction 0.4M Highest density fraction 2.5M Total gradient volume, incl. sample (mL) 12 Sample volume (mL) 6.5 Orientation Top-down Rotor type SW 40 Ti Speed (g) 192000 Duration (min) 1080 Fraction volume (mL) 0.5 Fraction processing Centrifugation Pelleting: volume per fraction 1.5 Pelleting: duration (min) 65 Pelleting: rotor type SW 40 Ti Pelleting: speed (g) 100000 EV-subtype Distinction between multiple subtypes Density Used subtypes 1.20-1.24 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Detected EV-associated proteins Flotillin1/ CD9/ CD63/ MFG-E8/ TSG101 Not detected contaminants beta-casein/ beta-lactoglobulin Characterization: RNA analysis RNA analysis Type (RT)(q)PCR Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis Particle analysis: flow cytometry Flow cytometer type BD Influx cell sorter (BD Biosciences, San Jose, CA, USA) Hardware adjustment High-resolution flow cytometric analysis of PKH67-stained samples was performed on a BD Influx cell sorter (BD Biosciences, San Jose, CA, USA) that was dedicated and optimized for detection of submicron-sized particles Calibration bead size 0.1 EV concentration Yes EM EM-type Cryo-EM Image type Close-up, Wide-field Report size (nm) 200

	EV200099	8/8	Bos taurus	milk	(d)(U)C DG	Kleinjan, Marije	2021	100%
Study summary Full title Regular Industrial Processing of Bovine Milk Impacts the Integrity and Molecular Composition of Extracellular Vesicles All authors Marije Kleinjan, Martijn Jc van Herwijnen, Sten Fwm Libregts, Rj Joost van Neerven, Anouk L Feitsma, Marca Hm Wauben Journal J Nutr Abstract Background: Bovine milk contains extracellular vesicles (EVs), which act as mediators of intercellul (show more...)Background: Bovine milk contains extracellular vesicles (EVs), which act as mediators of intercellular communication by regulating the recipients’ cellular processes via their selectively incorporated bioactive molecules. Because some of these EV components are evolutionarily conserved, EVs present in commercial milk might have the potential to regulate cellular processes in human consumers. Objectives: Because commercial milk is subjected to industrial processing, we investigated its effect on the number and integrity of isolated milk EVs and their bioactive components. For this, we compared EVs isolated from raw bovine milk with EVs isolated from different types of commercial milk, including pasteurized milk, either homogenized or not, and ultra heat treated (UHT) milk. Methods: EVs were separated from other milk components by differential centrifugation, followed by density gradient ultracentrifugation. EVs from different milk types were compared by single-particle high-resolution fluorescence-based flow cytometry to determine EV numbers, Cryo-electron microscopy to visualize EV integrity and morphology, western blot analysis to investigate EV-associated protein cargo, and RNA analysis to assess total small RNA concentration and milk-EV-specific microRNA expression. Results: In UHT milk, we could not detect intact EVs. Interestingly, although pasteurization (irrespective of homogenization) did not affect mean ± SD EV numbers (3.4 × 108 ± 1.2 × 108–2.8 × 108 ± 0.3 × 107 compared with 3.1 × 108 ± 1.2 × 108 in raw milk), it affected EV integrity and appearance, altered their protein signature, and resulted in a loss of milk-EV-associated RNAs (from 40.2 ± 3.4 ng/μL in raw milk to 17.7 ± 5.4–23.3 ± 10.0 mg/μL in processed milk, P < 0.05). Conclusions: Commercial milk, that has been heated by either pasteurization or UHT, contains fewer or no intact EVs, respectively. Although most EVs seemed resistant to pasteurization based on particle numbers, their integrity was affected and their molecular composition was altered. Thus, the possible transfer of bioactive components via bovine milk EVs to human consumers is likely diminished or altered in heat-treated commercial milk. (hide) EV-METRIC 100% (93rd percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type milk Sample origin UHT Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C DG Protein markers EV: MFG-E8/ CD63/ TSG101/ Flotillin1/ CD9 non-EV: beta-lactoglobulin/ beta-casein Proteomics no EV density (g/ml) 1.18-1.13 Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Bos taurus Sample Type milk Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Pelleting performed No Density gradient Type Continuous Lowest density fraction 0.4M Highest density fraction 2.5M Total gradient volume, incl. sample (mL) 12 Sample volume (mL) 6.5 Orientation Top-down Rotor type SW 40 Ti Speed (g) 192000 Duration (min) 1080 Fraction volume (mL) 0.5 Fraction processing Centrifugation Pelleting: volume per fraction 1.5 Pelleting: duration (min) 65 Pelleting: rotor type SW 40 Ti Pelleting: speed (g) 100000 EV-subtype Distinction between multiple subtypes Density Used subtypes 1.18-1.13 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Detected EV-associated proteins Flotillin1/ CD9/ CD63/ MFG-E8/ TSG101 Not detected contaminants beta-casein/ beta-lactoglobulin Characterization: RNA analysis RNA analysis Type (RT)(q)PCR Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis Particle analysis: flow cytometry Flow cytometer type BD Influx cell sorter (BD Biosciences, San Jose, CA, USA) Hardware adjustment High-resolution flow cytometric analysis of PKH67-stained samples was performed on a BD Influx cell sorter (BD Biosciences, San Jose, CA, USA) that was dedicated and optimized for detection of submicron-sized particles Calibration bead size 0.1 Report type Not Reported EV concentration Yes EM EM-type Cryo-EM Image type Close-up, Wide-field Report size (nm) 200

	EV200049	1/2	Homo sapiens	placental tissue culture supernatrant	DG (d)(U)C	Bergamelli, Mathilde	2021	100%
Study summary Full title Human Cytomegalovirus Infection Changes the Pattern of Surface Markers of Small Extracellular Vesicles Isolated From First Trimester Placental Long-Term Histocultures All authors Mathilde Bergamelli, Hélène Martin, Mélinda Bénard, Jérôme Ausseil, Jean-Michel Mansuy, Ilse Hurbain, Maïlys Mouysset, Marion Groussolles, Géraldine Cartron, Yann Tanguy le Gac, Nathalie Moinard, Elsa Suberbielle, Jacques Izopet, Charlotte Tscherning, Graça Raposo, Daniel Gonzalez-Dunia, Gisela D'Angelo, Cécile E Malnou Journal Front Cell Dev Biol Abstract Extracellular vesicles (EVs) have increasingly been recognized as key players in a wide variety of p (show more...)Extracellular vesicles (EVs) have increasingly been recognized as key players in a wide variety of physiological and pathological contexts, including during pregnancy. Notably, EVs appear both as possible biomarkers and as mediators involved in the communication of the placenta with the maternal and fetal sides. A better understanding of the physiological and pathological roles of EVs strongly depends on the development of adequate and reliable study models, specifically at the beginning of pregnancy where many adverse pregnancy outcomes have their origin. In this study, we describe the isolation of small EVs from a histoculture model of first trimester placental explants in normal conditions as well as upon infection by human cytomegalovirus. Using bead-based multiplex cytometry and electron microscopy combined with biochemical approaches, we characterized these small EVs and defined their associated markers and ultrastructure. We observed that infection led to changes in the expression level of several surface markers, without affecting the secretion and integrity of small EVs. Our findings lay the foundation for studying the functional role of EVs during early pregnancy, along with the identification of new predictive biomarkers for the severity and outcome of this congenital infection, which are still sorely lacking. (hide) EV-METRIC 100% (50th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type placental tissue culture supernatrant Sample origin Control condition Focus vesicles Other / small extracellular vesicles Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture DG (d)(U)C Protein markers EV: CD29/ CD63/ CD81/ CD44/ CD326/ CD9 non-EV: Calreticulin Proteomics no EV density (g/ml) 1.103 Show all info Study aim New methodological development Sample Species Homo sapiens Sample Type placental tissue culture supernatrant Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 60 Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 100000 Density gradient Type Discontinuous Number of initial discontinuous layers 3 Lowest density fraction 10% Highest density fraction 40% Total gradient volume, incl. sample (mL) 10 Sample volume (mL) 1 Orientation Bottom-up Rotor type SW 41 Ti Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 1.7 Fraction processing Centrifugation Pelleting: volume per fraction 25 Pelleting: duration (min) 60 Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Detected EV-associated proteins CD63 Not detected EV-associated proteins CD81/ CD9 Not detected contaminants Calreticulin Detected EV-associated proteins CD9/ CD29/ CD44/ CD326/ CD81/ CD63 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Modus Reported size (nm) 143 EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 1.00E+08 Particle analysis: flow cytometry Flow cytometer type mascquant VYB Hardware adjustment Use of calibration beads FITC positive, of different size (500, 240, 200 and 160 nm) and granulosity that allow bead population separation and calibration of the cytometer , creation of a gate on 200 nm and smaller events Calibration bead size 0.16 EV concentration Yes EM EM-type Immuno-EM/ Transmission-EM EM protein CD63 Image type Close-up, Wide-field Report size (nm) 95

	EV200049	2/2	Homo sapiens	placental tissue culture supernatrant	DG (d)(U)C	Bergamelli, Mathilde	2021	100%
Study summary Full title Human Cytomegalovirus Infection Changes the Pattern of Surface Markers of Small Extracellular Vesicles Isolated From First Trimester Placental Long-Term Histocultures All authors Mathilde Bergamelli, Hélène Martin, Mélinda Bénard, Jérôme Ausseil, Jean-Michel Mansuy, Ilse Hurbain, Maïlys Mouysset, Marion Groussolles, Géraldine Cartron, Yann Tanguy le Gac, Nathalie Moinard, Elsa Suberbielle, Jacques Izopet, Charlotte Tscherning, Graça Raposo, Daniel Gonzalez-Dunia, Gisela D'Angelo, Cécile E Malnou Journal Front Cell Dev Biol Abstract Extracellular vesicles (EVs) have increasingly been recognized as key players in a wide variety of p (show more...)Extracellular vesicles (EVs) have increasingly been recognized as key players in a wide variety of physiological and pathological contexts, including during pregnancy. Notably, EVs appear both as possible biomarkers and as mediators involved in the communication of the placenta with the maternal and fetal sides. A better understanding of the physiological and pathological roles of EVs strongly depends on the development of adequate and reliable study models, specifically at the beginning of pregnancy where many adverse pregnancy outcomes have their origin. In this study, we describe the isolation of small EVs from a histoculture model of first trimester placental explants in normal conditions as well as upon infection by human cytomegalovirus. Using bead-based multiplex cytometry and electron microscopy combined with biochemical approaches, we characterized these small EVs and defined their associated markers and ultrastructure. We observed that infection led to changes in the expression level of several surface markers, without affecting the secretion and integrity of small EVs. Our findings lay the foundation for studying the functional role of EVs during early pregnancy, along with the identification of new predictive biomarkers for the severity and outcome of this congenital infection, which are still sorely lacking. (hide) EV-METRIC 100% (50th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type placental tissue culture supernatrant Sample origin in vitro hCMV infected placental tissue Focus vesicles Other / small extracellular vesicles Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture DG (d)(U)C Protein markers EV: CD29/ CD63/ CD81/ CD44/ CD326/ CD9 non-EV: Calreticulin Proteomics no EV density (g/ml) 1.103 Show all info Study aim New methodological development Sample Species Homo sapiens Sample Type placental tissue culture supernatrant Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 60 Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 100000 Density gradient Type Discontinuous Number of initial discontinuous layers 3 Lowest density fraction 10% Highest density fraction 40% Total gradient volume, incl. sample (mL) 10 Sample volume (mL) 1 Orientation Bottom-up Rotor type SW 41 Ti Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 1.7 Fraction processing Centrifugation Pelleting: volume per fraction 25 Pelleting: duration (min) 60 Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Detected EV-associated proteins CD63 Not detected EV-associated proteins CD63/ CD9 Not detected contaminants Calreticulin Detected EV-associated proteins CD9/ CD29/ CD44/ CD326/ CD81/ CD63 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Not Reported Particle analysis: flow cytometry Flow cytometer type mascquant VYB Hardware adjustment Use of calibration beads FITC positive, of different size (500, 240, 200 and 160 nm) and granulosity that allow bead population separation and calibration of the cytometer , creation of a gate on 200 nm and smaller events Calibration bead size 0.16 Report type Mean Reported size (nm) 140 EV concentration Yes EM EM-type Immuno-EM/ Transmission-EM EM protein CD63 Image type Close-up, Wide-field Report size (nm) 100

	EV200010	4/4	Homo sapiens	Blood plasma	DG (d)(U)C SEC	Kuypers, Sören	2021	100%
Study summary Full title Unsupervised Machine Learning-Based Clustering of Nanosized Fluorescent Extracellular Vesicles All authors Sören Kuypers, Nick Smisdom, Isabel Pintelon, Jean-Pierre Timmermans, Marcel Ameloot, Luc Michiels, Jelle Hendrix, Baharak Hosseinkhani Journal Small Abstract Extracellular vesicles (EV) are biological nanoparticles that play an important role in cell-to-cell (show more...)Extracellular vesicles (EV) are biological nanoparticles that play an important role in cell-to-cell communication. The phenotypic profile of EV populations is a promising reporter of disease, with direct clinical diagnostic relevance. Yet, robust methods for quantifying the biomarker content of EV have been critically lacking, and require a single-particle approach due to their inherent heterogeneous nature. Here, multicolor single-molecule burst analysis microscopy is used to detect multiple biomarkers present on single EV. The authors classify the recorded signals and apply the machine learning-based t-distributed stochastic neighbor embedding algorithm to cluster the resulting multidimensional data. As a proof of principle, the authors use the method to assess both the purity and the inflammatory status of EV, and compare cell culture and plasma-derived EV isolated via different purification methods. This methodology is then applied to identify intercellular adhesion molecule-1 specific EV subgroups released by inflamed endothelial cells, and to prove that apolipoprotein-a1 is an excellent marker to identify the typical lipoprotein contamination in plasma. This methodology can be widely applied on standard confocal microscopes, thereby allowing both standardized quality assessment of patient plasma EV preparations, and diagnostic profiling of multiple EV biomarkers in health and disease. (hide) EV-METRIC 100% (99th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Blood plasma Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture DG (d)(U)C SEC Protein markers EV: ICAM/ CD63/ CD9/ ANXA2 non-EV: APOA1 Proteomics no EV density (g/ml) 1.1 Show all info Study aim New methodological development/Biomarker/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Blood plasma Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Pelleting performed No Density gradient Only used for validation of main results Yes Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 16.5 Sample volume (mL) 1 Orientation Top-down Rotor type SW 28.1 Speed (g) 100000 Duration (min) 1451 Fraction volume (mL) 1 Fraction processing Size-exclusion chromatography Size-exclusion chromatography Total column volume (mL) 10 Sample volume/column (mL) 1-6 Resin type Sepharose CL-2B Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Detected EV-associated proteins CD9/ ANXA2 Not detected contaminants APOA1 Detected EV-associated proteins CD9/ CD63/ ICAM Not detected contaminants APOA1 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 100-200 EV concentration Yes EM EM-type Transmission-EM Image type Close-up, Wide-field Report size (nm) 100-200

	EV190096	1/2	Bos taurus	skim milk	acetic acid treatment DG (d)(U)C Filtration	Mukhopadhya, Anindya	2021	100%
Study summary Full title Optimisation and comparison of orthogonal methods for separation and characterisation of extracellular vesicles to investigate how representative infant milk formula is of milk All authors Anindya Mukhopadhya, Jessie Santoro, Barry Moran, Zivile Useckaite, Lorraine O'Driscoll Journal Food Chem. Abstract Many infants are fed infant milk formula (IMF). However, IMF production from skim milk (SM) involves (show more...)Many infants are fed infant milk formula (IMF). However, IMF production from skim milk (SM) involves harsh treatment. So, we hypothesised that the quantity and/or quality of extracellular vesicles (EVs) in IMF may be reduced. Thus, firstly, we aimed to optimise separation of EVs from IMF and SM and, secondly, we aimed to compare the EV isolates from these two sources. Prior to EV isolation, abundant casein micelles of similar sizes to EVs were removed by treating milk samples with either acetic acid or hydrochloric acid. Samples progressed to differential ultracentrifugation (DUC) or gradient ultracentrifugation (GUC). EV characterisation included BCA, SDS-PAGE, nanoparticle tracking (NTA), electron microscopy (TEM), immunoblotting, and imaging flow cytometry (IFCM). Reduced EV concentrations were found in IMF. SM-derived EVs were intact, while IMF contained disrupted EV-like structures. EV biomarkers were more abundant with isolates from SM, indicating EV proteins in IMF are compromised. Altogether, a suitable method combining acid pre-treatment with GUC for EV separation from milk products was developed. EVs appear to be substantially compromised in IMF compared to SM. (hide) EV-METRIC 100% (50th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type skim milk Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture acetic acid treatment DG (d)(U)C Filtration Protein markers EV: TSG101/ CD63/ CD81/ HLADR/ ADAM10/ CD9 non-EV: Actinin4 Proteomics no EV density (g/ml) 1.15 Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Bos taurus Sample Type skim milk Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Equal to or above 150,000 g Between 50,000 g and 100,000 g Pelleting performed No Density gradient Type Discontinuous Number of initial discontinuous layers 5 Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 17 Sample volume (mL) 2.33 Orientation Bottom-up Rotor type Type 70.1Ti Speed (g) 186000 Duration (min) 1080 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 9 Pelleting: duration (min) 90 Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 110000 Filtration steps 0.45µm > x > 0.22µm, Other Name other separation method acetic acid treatment Other Name other separation method Characterization: Protein analysis Protein Concentration Method BCA Western Blot Detected EV-associated proteins CD63/ TSG101 Not detected EV-associated proteins Detected contaminants Not detected contaminants Actinin4 Flow cytometry Type of Flow cytometry AMNIS ImageStreamX Mark II Flow Cytometer Calibration bead size none Detected EV-associated proteins CD63/ CD9/ CD81/ ADAM10/ HLADR Not detected EV-associated proteins Detected contaminants Not detected contaminants Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 160 EV concentration Yes

	EV190096	2/2	Bos taurus	powdered infant milk formula	acetic acid treatment DG (d)(U)C Filtration	Mukhopadhya, Anindya	2021	100%
Study summary Full title Optimisation and comparison of orthogonal methods for separation and characterisation of extracellular vesicles to investigate how representative infant milk formula is of milk All authors Anindya Mukhopadhya, Jessie Santoro, Barry Moran, Zivile Useckaite, Lorraine O'Driscoll Journal Food Chem. Abstract Many infants are fed infant milk formula (IMF). However, IMF production from skim milk (SM) involves (show more...)Many infants are fed infant milk formula (IMF). However, IMF production from skim milk (SM) involves harsh treatment. So, we hypothesised that the quantity and/or quality of extracellular vesicles (EVs) in IMF may be reduced. Thus, firstly, we aimed to optimise separation of EVs from IMF and SM and, secondly, we aimed to compare the EV isolates from these two sources. Prior to EV isolation, abundant casein micelles of similar sizes to EVs were removed by treating milk samples with either acetic acid or hydrochloric acid. Samples progressed to differential ultracentrifugation (DUC) or gradient ultracentrifugation (GUC). EV characterisation included BCA, SDS-PAGE, nanoparticle tracking (NTA), electron microscopy (TEM), immunoblotting, and imaging flow cytometry (IFCM). Reduced EV concentrations were found in IMF. SM-derived EVs were intact, while IMF contained disrupted EV-like structures. EV biomarkers were more abundant with isolates from SM, indicating EV proteins in IMF are compromised. Altogether, a suitable method combining acid pre-treatment with GUC for EV separation from milk products was developed. EVs appear to be substantially compromised in IMF compared to SM. (hide) EV-METRIC 100% (50th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type powdered infant milk formula Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture acetic acid treatment DG (d)(U)C Filtration Protein markers EV: TSG101/ CD63/ CD81/ HLADR/ ADAM10/ CD9 non-EV: Actinin4 Proteomics no EV density (g/ml) 1.15 Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Bos taurus Sample Type powdered infant milk formula Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Equal to or above 150,000 g Between 50,000 g and 100,000 g Pelleting performed No Density gradient Type Discontinuous Number of initial discontinuous layers 5 Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 17 Sample volume (mL) 2.33 Orientation Bottom-up Rotor type Type 70.1Ti Speed (g) 186000 Duration (min) 1080 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 9 Pelleting: duration (min) 90 Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 110000 Filtration steps 0.45µm > x > 0.22µm, Other Name other separation method acetic acid treatment Other Name other separation method Characterization: Protein analysis Protein Concentration Method BCA Western Blot Detected EV-associated proteins CD63/ TSG101 Not detected EV-associated proteins Detected contaminants Not detected contaminants Actinin4 Flow cytometry Type of Flow cytometry AMNIS ImageStreamX Mark II Flow Cytometer Calibration bead size none Detected EV-associated proteins CD63/ CD9/ CD81/ ADAM10/ HLADR Not detected EV-associated proteins Detected contaminants Not detected contaminants Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 175 EV concentration Yes

	EV210262	1/2	Homo sapiens	hiPSC-derived RPE	(d)(U)C DG Filtration	Flores-Bellver, Miguel	2021	89%
Study summary Full title Extracellular vesicles released by human retinal pigment epithelium mediate increased polarised secretion of drusen proteins in response to AMD stressors All authors Miguel Flores-Bellver, Jason Mighty, Silvia Aparicio-Domingo, Kang V Li, Cui Shi, Jing Zhou, Hannah Cobb, Patrick McGrath, German Michelis, Patricia Lenhart, Ganna Bilousova, Søren Heissel, Michael J Rudy, Christina Coughlan 10 , Andrew E Goodspeed 11 12 , S Patricia Becerra, Stephen Redenti 13 , M Valeria Canto-Soler Journal J Extracell Vesicles Abstract Age-related macular degeneration (AMD) is a leading cause of blindness worldwide. Drusen are key con (show more...)Age-related macular degeneration (AMD) is a leading cause of blindness worldwide. Drusen are key contributors to the etiology of AMD and the ability to modulate drusen biogenesis could lead to therapeutic strategies to slow or halt AMD progression. The mechanisms underlying drusen biogenesis, however, remain mostly unknown. Here we demonstrate that under homeostatic conditions extracellular vesicles (EVs) secreted by retinal pigment epithelium (RPE) cells are enriched in proteins associated with mechanisms involved in AMD pathophysiology, including oxidative stress, immune response, inflammation, complement system and drusen composition. Furthermore, we provide first evidence that drusen-associated proteins are released as cargo of extracellular vesicles secreted by RPE cells in a polarised apical:basal mode. Notably, drusen-associated proteins exhibited distinctive directional secretion modes in homeostatic conditions and, differential modulation of this directional secretion in response to AMD stressors. These observations underpin the existence of a finely-tuned mechanism regulating directional apical:basal sorting and secretion of drusen-associated proteins via EVs, and its modulation in response to mechanisms involved in AMD pathophysiology. Collectively, our results strongly support an active role of RPE-derived EVs as a key source of drusen proteins and important contributors to drusen development and growth. (hide) EV-METRIC 89% (99th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Density gradient Filtration Adj. k-factor 7.66 (washing) Protein markers EV: CD63/ Flotillin-1/ HSP70/ HSP90/ TSG101 non-EV: GM130 Proteomics yes EV density (g/ml) 1.064 Show all info Study aim Mechanism of uptake/transfer/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells hiPSC-derived RPE EV-harvesting Medium EV-depleted medium Preparation of EDS Commercial EDS Cell viability (%) 99 Cell count 8400000 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 90 Pelleting: rotor type Type 70.1Ti Pelleting: speed (g) 120000 Wash: volume per pellet (ml) 10 Wash: time (min) 90 Wash: Rotor Type Type 70.1Ti Wash: speed (g) 120000 Wash: adjusted k-factor 7.66E Density gradient Only used for validation of main results Yes Type Continuous Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 10 Sample volume (mL) 1 Orientation Top-down Rotor type SW 41 Ti Speed (g) 141000 Duration (min) 3600 Fraction volume (mL) 0.5 Fraction processing Centrifugation Pelleting: volume per fraction 9 Pelleting: speed (g) 120000 Pelleting: adjusted k-factor 7.66E Filtration steps 0.2 or 0.22 ?m Characterization: Protein analysis Protein Concentration Method microBCA Protein Yield (µg) 15 Western Blot Detected EV-associated proteins CD63/ Flotillin-1/ HSP70/ HSP90/ TSG101 Detected contaminants GM130 Proteomics database Yes Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 120 EV concentration Yes Particle yield particles per milliliter of starting sample: 4.00e+10 EM EM-type Transmission-EM Image type Close-up Report size (nm) 120

	EV210262	2/2	Homo sapiens	hiPSC-derived RPE	(d)(U)C DG Filtration	Flores-Bellver, Miguel	2021	89%
Study summary Full title Extracellular vesicles released by human retinal pigment epithelium mediate increased polarised secretion of drusen proteins in response to AMD stressors All authors Miguel Flores-Bellver, Jason Mighty, Silvia Aparicio-Domingo, Kang V Li, Cui Shi, Jing Zhou, Hannah Cobb, Patrick McGrath, German Michelis, Patricia Lenhart, Ganna Bilousova, Søren Heissel, Michael J Rudy, Christina Coughlan 10 , Andrew E Goodspeed 11 12 , S Patricia Becerra, Stephen Redenti 13 , M Valeria Canto-Soler Journal J Extracell Vesicles Abstract Age-related macular degeneration (AMD) is a leading cause of blindness worldwide. Drusen are key con (show more...)Age-related macular degeneration (AMD) is a leading cause of blindness worldwide. Drusen are key contributors to the etiology of AMD and the ability to modulate drusen biogenesis could lead to therapeutic strategies to slow or halt AMD progression. The mechanisms underlying drusen biogenesis, however, remain mostly unknown. Here we demonstrate that under homeostatic conditions extracellular vesicles (EVs) secreted by retinal pigment epithelium (RPE) cells are enriched in proteins associated with mechanisms involved in AMD pathophysiology, including oxidative stress, immune response, inflammation, complement system and drusen composition. Furthermore, we provide first evidence that drusen-associated proteins are released as cargo of extracellular vesicles secreted by RPE cells in a polarised apical:basal mode. Notably, drusen-associated proteins exhibited distinctive directional secretion modes in homeostatic conditions and, differential modulation of this directional secretion in response to AMD stressors. These observations underpin the existence of a finely-tuned mechanism regulating directional apical:basal sorting and secretion of drusen-associated proteins via EVs, and its modulation in response to mechanisms involved in AMD pathophysiology. Collectively, our results strongly support an active role of RPE-derived EVs as a key source of drusen proteins and important contributors to drusen development and growth. (hide) EV-METRIC 89% (99th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Cigarette smoke treatment Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Density gradient Filtration Adj. k-factor 7.66 (washing) Protein markers EV: CD63/ Flotillin-1/ HSP70/ HSP90/ TSG101 non-EV: GM130 Proteomics yes EV density (g/ml) 1.064 Show all info Study aim Mechanism of uptake/transfer/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells hiPSC-derived RPE EV-harvesting Medium EV-depleted medium Preparation of EDS Commercial EDS Cell viability (%) 99 Cell count 8400000 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 90 Pelleting: rotor type Type 70.1Ti Pelleting: speed (g) 120000 Wash: volume per pellet (ml) 10 Wash: time (min) 90 Wash: Rotor Type Type 70.1Ti Wash: speed (g) 120000 Wash: adjusted k-factor 7.66E Density gradient Only used for validation of main results Yes Type Continuous Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 10 Sample volume (mL) 1 Orientation Top-down Rotor type SW 41 Ti Speed (g) 141000 Duration (min) 3600 Fraction volume (mL) 0.5 Fraction processing Centrifugation Pelleting: volume per fraction 9 Pelleting: speed (g) 120000 Pelleting: adjusted k-factor 7.66E Filtration steps 0.2 or 0.22 ?m Characterization: Protein analysis Protein Concentration Method microBCA Protein Yield (µg) 15 Western Blot Detected EV-associated proteins CD63/ Flotillin-1/ HSP70/ HSP90/ TSG101 Detected contaminants GM130 Proteomics database Yes Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 120 EV concentration Yes Particle yield particles per milliliter of starting sample: 4.70e+11 EM EM-type Transmission-EM Image type Close-up Report size (nm) 120

	EV210261	1/8	Homo sapiens	SW620	(d)(U)C DG	Rai, Alin	2021	89%
Study summary Full title Proteomic dissection of large extracellular vesicle surfaceome unravels interactive surface platform All authors Alin Rai, Haoyun Fang, Bethany Claridge, Richard J. Simpson, and David W Greening Journal J Extracell Vesicles Abstract The extracellular vesicle (EV) surface proteome (surfaceome) acts as a fundamental signalling gatewa (show more...)The extracellular vesicle (EV) surface proteome (surfaceome) acts as a fundamental signalling gateway by bridging intra‐ and extracellular signalling networks, dictates EVs’ capacity to communicate and interact with their environment, and is a source of potential disease biomarkers and therapeutic targets. However, our understanding of surface protein composition of large EVs (L‐EVs, 100–800 nm, mean 310 nm, ATP5F1A, ATP5F1B, DHX9, GOT2, HSPA5, HSPD1, MDH2, STOML2), a major EV‐subtype that are distinct from small EVs (S‐EVs, 30–150 nm, mean 110 nm, CD44, CD63, CD81, CD82, CD9, PDCD6IP, SDCBP, TSG101) remains limited. Using a membrane impermeant derivative of biotin to capture surface proteins coupled to mass spectrometry analysis, we show that out of 4143 proteins identified in density‐gradient purified L‐EVs (1.07–1.11 g/mL, from multiple cancer cell lines), 961 proteins are surface accessible. The surface molecular diversity of L‐EVs include (i) bona fide plasma membrane anchored proteins (cluster of differentiation, transporters, receptors and GPI anchored proteins implicated in cell‐cell and cell‐ECM interactions); and (ii) membrane surface‐associated proteins (that are released by divalent ion chelator EDTA) implicated in actin cytoskeleton regulation, junction organization, glycolysis and platelet activation. Ligand‐receptor analysis of L‐EV surfaceome (e.g., ITGAV/ITGB1) uncovered interactome spanning 172 experimentally verified cognate binding partners (e.g., ANGPTL3, PLG, and VTN) with highest tissue enrichment for liver. Assessment of biotin inaccessible L‐EV proteome revealed enrichment for proteins belonging to COPI/II‐coated ER/Golgi‐derived vesicles and mitochondria. Additionally, despite common surface proteins identified in L‐EVs and S‐EVs, our data reveals surfaceome heterogeneity between the two EV‐subtype. Collectively, our study provides critical insights into diverse proteins operating at the interactive platform of L‐EVs and molecular leads for future studies seeking to decipher L‐EV heterogeneity and function. (hide) EV-METRIC 89% (99th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Density gradient Protein markers EV: Alix/ CD63/ TSG101 non-EV: None Proteomics yes EV density (g/ml) 1.07-1.11 Show all info Study aim Identification of content (omics approaches)/Technical analysis comparing/optimizing EV-?related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells SW620 EV-harvesting Medium Serum free medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 10,000 g and 50,000 g Pelleting performed Yes Pelleting: time(min) 30 Pelleting: rotor type SW 28 Pelleting: speed (g) 10000 Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 12 Sample volume (mL) 0.5 Orientation Top-down Rotor type SW 41 Ti Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 2 Pelleting: speed (g) 10000 Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Detected EV-associated proteins Alix/ CD63/ TSG101 Proteomics database Yes Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type mean and size range/distribution Reported size (nm) 166 mean, range 50-250 nm EM EM-type Cryo-EM Image type Close-up, Wide-field

	EV210261	2/8	Homo sapiens	SW620	(d)(U)C DG	Rai, Alin	2021	89%
Study summary Full title Proteomic dissection of large extracellular vesicle surfaceome unravels interactive surface platform All authors Alin Rai, Haoyun Fang, Bethany Claridge, Richard J. Simpson, and David W Greening Journal J Extracell Vesicles Abstract The extracellular vesicle (EV) surface proteome (surfaceome) acts as a fundamental signalling gatewa (show more...)The extracellular vesicle (EV) surface proteome (surfaceome) acts as a fundamental signalling gateway by bridging intra‐ and extracellular signalling networks, dictates EVs’ capacity to communicate and interact with their environment, and is a source of potential disease biomarkers and therapeutic targets. However, our understanding of surface protein composition of large EVs (L‐EVs, 100–800 nm, mean 310 nm, ATP5F1A, ATP5F1B, DHX9, GOT2, HSPA5, HSPD1, MDH2, STOML2), a major EV‐subtype that are distinct from small EVs (S‐EVs, 30–150 nm, mean 110 nm, CD44, CD63, CD81, CD82, CD9, PDCD6IP, SDCBP, TSG101) remains limited. Using a membrane impermeant derivative of biotin to capture surface proteins coupled to mass spectrometry analysis, we show that out of 4143 proteins identified in density‐gradient purified L‐EVs (1.07–1.11 g/mL, from multiple cancer cell lines), 961 proteins are surface accessible. The surface molecular diversity of L‐EVs include (i) bona fide plasma membrane anchored proteins (cluster of differentiation, transporters, receptors and GPI anchored proteins implicated in cell‐cell and cell‐ECM interactions); and (ii) membrane surface‐associated proteins (that are released by divalent ion chelator EDTA) implicated in actin cytoskeleton regulation, junction organization, glycolysis and platelet activation. Ligand‐receptor analysis of L‐EV surfaceome (e.g., ITGAV/ITGB1) uncovered interactome spanning 172 experimentally verified cognate binding partners (e.g., ANGPTL3, PLG, and VTN) with highest tissue enrichment for liver. Assessment of biotin inaccessible L‐EV proteome revealed enrichment for proteins belonging to COPI/II‐coated ER/Golgi‐derived vesicles and mitochondria. Additionally, despite common surface proteins identified in L‐EVs and S‐EVs, our data reveals surfaceome heterogeneity between the two EV‐subtype. Collectively, our study provides critical insights into diverse proteins operating at the interactive platform of L‐EVs and molecular leads for future studies seeking to decipher L‐EV heterogeneity and function. (hide) EV-METRIC 89% (99th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Density gradient Protein markers EV: CD63 non-EV: None Proteomics yes EV density (g/ml) 1.07-1.11 Show all info Study aim Identification of content (omics approaches)/Technical analysis comparing/optimizing EV-?related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells SW620 EV-harvesting Medium Serum free medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 60 Pelleting: rotor type SW 41 Ti Pelleting: speed (g) 100000 Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 12 Sample volume (mL) 0.5 Orientation Top-down Rotor type SW 41 Ti Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 2 Pelleting: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Detected EV-associated proteins CD63 Proteomics database Yes Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type mean and size range/distribution Reported size (nm) 50-500 EM EM-type Cryo-EM Image type Close-up, Wide-field Report size (nm) 310

	EV210043	2/2	Homo sapiens	hcMEC/D3	DG (d)(U)C UF Filtration	Fikatas, Antonios	2021	89%
Study summary Full title Deciphering the Role of Extracellular Vesicles Derived from ZIKV-Infected hcMEC/D3 Cells on the Blood-Brain Barrier System All authors Antonios Fikatas, Jonas Dehairs, Sam Noppen, Jordi Doijen, Frank Vanderhoydonc, Eef Meyen, Johannes V Swinnen, Christophe Pannecouque, Dominique Schols Journal Viruses Abstract To date, no vaccines or antivirals are available against Zika virus (ZIKV). In addition, the mechani (show more...)To date, no vaccines or antivirals are available against Zika virus (ZIKV). In addition, the mechanisms underlying ZIKV-associated pathogenesis of the central nervous system (CNS) are largely unexplored. Getting more insight into the cellular pathways that ZIKV recruits to facilitate infection of susceptible cells will be crucial for establishing an effective treatment strategy. In general, cells secrete a number of vesicles, known as extracellular vesicles (EVs), in response to viral infections. These EVs serve as intercellular communicators. Here, we investigated the role of EVs derived from ZIKV-infected human brain microvascular endothelial cells on the blood–brain barrier (BBB) system. We demonstrated that ZIKV-infected EVs (IEVs) can incorporate viral components, including ZIKV RNA, NS1, and E-protein, and further transfer them to several types of CNS cells. Using label-free impedance-based biosensing, we observed that ZIKV and IEVs can temporally disturb the monolayer integrity of BBB-mimicking cells, possibly by inducing structural rearrangements of the adherent protein VE-cadherin (immunofluorescence staining). Finally, differences in the lipidomic profile between EVs and their parental cells possibly suggest a preferential sorting mechanism of specific lipid species into the vesicles. To conclude, these data suggest that IEVs could be postulated as vehicles (Trojan horse) for ZIKV transmission via the BBB. (hide) EV-METRIC 89% (99th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin ZIKV-infected Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture DG (d)(U)C UF Filtration Protein markers EV: TSG101/ Alix/ CD63 non-EV: Calnexin Proteomics no EV density (g/ml) 1.06-1.16 Show all info Study aim Function/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells hcMEC/D3 EV-harvesting Medium EV-depleted medium Preparation of EDS Commercial EDS Cell viability (%) NA Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Pelleting performed Yes Pelleting: time(min) 1260 Pelleting: rotor type TH-641 Pelleting: speed (g) 100 000 Wash: volume per pellet (ml) 10 Wash: time (min) 180 Wash: Rotor Type TH-641 Wash: speed (g) 100 000 Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 10 Sample volume (mL) 1 Orientation Top-down Rotor type TH-641 Speed (g) 100 000 Duration (min) 1080 Fraction volume (mL) 1,5 Fraction processing None Filtration steps 0.22µm or 0.2µm Ultra filtration Cut-off size (kDa) 10 Membrane type Regenerated cellulose Characterization: Protein analysis Protein Concentration Method Bradford Western Blot Detected EV-associated proteins CD63/ TSG101/ Alix Not detected contaminants Calnexin Characterization: Lipid analysis Yes Characterization: Particle analysis NTA Report type Mean Reported size (nm) 175 EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 1,40E+08 EM EM-type Transmission-EM Image type Wide-field Report size (nm) 130

	EV210021	1/2	Rattus norvegicus	Primary rat hepatocytes	(d)(U)C Filtration DG	Mleczko, J.E.	2021	89%
Study summary Full title Extracellular vesicles released by steatotic hepatocytes alteradipocyte metabolism All authors J.E. Mleczko, F. Royo, I. Samuelson, M. Clos-Garcia, C. Williams, D. Cabrera, M. Azparren-Angulo, E. Gonzalez, C. Garcia-Vallicrosa, S. Carobbio, S. Rodriguez-Cuenca, M. Azkargorta, S. van Liempd, F. Elortza, A. Vidal-Puig, S. Mora, J.M. Falcon-Perez Journal Journal of Extracellular Biology Abstract The composition of extracellular vesicles (EVs) is altered in many pathological condi-tions, and the (show more...)The composition of extracellular vesicles (EVs) is altered in many pathological condi-tions, and their molecular content provides essential information on features of par-ent cells and mechanisms of crosstalk between cells and organs. Metabolic Syndrome(MetS) is a cluster of clinical manifestations including obesity, insulin resistance, dys-lipidemia and hypertension that increases the risk of cardiovascular disease and type 2diabetes mellitus. Here, we investigated the crosstalk between liver and adipocytes bycharacterizing EVs secreted by primary hepatocytes isolated from Zucker rat model,and studied the effect they have on 3T3-L1 adipocytes. We found that steatotic hepa-tocytes secrete EVs with significantly reduced exosomal markers in comparison withtheir lean counterpart. Moreover, proteomic analysis revealed that those EVs reflectthe metabolic state of the parent cell in that the majority of proteins upregulated relateto fat metabolism, fatty acid synthesis, glycolysis, and pentose phosphate pathway.In addition, hepatocytes-secreted EVs influenced lipolysis and insulin sensitivity inrecipient 3T3-L1 adipocytes. Untargeted metabolomic analysis detected alterations indifferent adipocyte metabolic pathways in cells treated with hepatic EVs. In summary,our work showed that steatosis has a significant impact in the amount and composi-tion of EVs secreted by hepatocytes. Moreover, our data point to the involvement ofhepatic-EVs in the development of pathologies associated with MetS. (hide) EV-METRIC 89% (99th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Obese hepatocytes Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Filtration Density gradient Protein markers EV: TSG101/ CD63/ CD81/ HSP90/ Alix/ Flotillin1/ HSP70 non-EV: GRP78/ COXIV/ Parp Proteomics yes Show all info Study aim Function/Identification of content (omics approaches) Sample Species Rattus norvegicus Sample Type Cell culture supernatant EV-producing cells Primary rat hepatocytes EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g Cell count 300000000 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Between 50,000 g and 100,000 g Pelleting performed Yes Pelleting: rotor type Type 45 Ti Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 45 Wash: time (min) 90 Wash: Rotor Type Type 45 Ti Wash: speed (g) 100000 Density gradient Only used for validation of main results Yes Type Discontinuous Number of initial discontinuous layers 2 Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 16,5 Sample volume (mL) 5,5 Orientation Top-down Speed (g) 100000 Duration (min) 160 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction Not repo Pelleting: duration (min) 60 Pelleting: rotor type Not reported Pelleting: speed (g) 100000 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method Bradford Western Blot Detected EV-associated proteins Flotillin1/ CD63/ HSP90/ TSG101/ HSP70/ Alix/ CD81 Not detected EV-associated proteins HSP90/ HSP70/ CD81/ Flotillin1/ TSG101/ CD63/ Alix Detected contaminants COXIV/ GRP78/ Parp Not detected contaminants COXIV/ GRP78/ Parp Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 50-400 EV concentration Yes Particle yield as number of particles per million cells: 2 EM EM-type Cryo-EM Image type Close-up

	EV210021	2/2	Rattus norvegicus	Primary rat hepatocytes	(d)(U)C Filtration	Mleczko, J.E.	2021	89%
Study summary Full title Extracellular vesicles released by steatotic hepatocytes alteradipocyte metabolism All authors J.E. Mleczko, F. Royo, I. Samuelson, M. Clos-Garcia, C. Williams, D. Cabrera, M. Azparren-Angulo, E. Gonzalez, C. Garcia-Vallicrosa, S. Carobbio, S. Rodriguez-Cuenca, M. Azkargorta, S. van Liempd, F. Elortza, A. Vidal-Puig, S. Mora, J.M. Falcon-Perez Journal Journal of Extracellular Biology Abstract The composition of extracellular vesicles (EVs) is altered in many pathological condi-tions, and the (show more...)The composition of extracellular vesicles (EVs) is altered in many pathological condi-tions, and their molecular content provides essential information on features of par-ent cells and mechanisms of crosstalk between cells and organs. Metabolic Syndrome(MetS) is a cluster of clinical manifestations including obesity, insulin resistance, dys-lipidemia and hypertension that increases the risk of cardiovascular disease and type 2diabetes mellitus. Here, we investigated the crosstalk between liver and adipocytes bycharacterizing EVs secreted by primary hepatocytes isolated from Zucker rat model,and studied the effect they have on 3T3-L1 adipocytes. We found that steatotic hepa-tocytes secrete EVs with significantly reduced exosomal markers in comparison withtheir lean counterpart. Moreover, proteomic analysis revealed that those EVs reflectthe metabolic state of the parent cell in that the majority of proteins upregulated relateto fat metabolism, fatty acid synthesis, glycolysis, and pentose phosphate pathway.In addition, hepatocytes-secreted EVs influenced lipolysis and insulin sensitivity inrecipient 3T3-L1 adipocytes. Untargeted metabolomic analysis detected alterations indifferent adipocyte metabolic pathways in cells treated with hepatic EVs. In summary,our work showed that steatosis has a significant impact in the amount and composi-tion of EVs secreted by hepatocytes. Moreover, our data point to the involvement ofhepatic-EVs in the development of pathologies associated with MetS. (hide) EV-METRIC 89% (99th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Lean hepatocytes Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Filtration Protein markers EV: TSG101/ CD63/ CD81/ HSP90/ Alix/ Flotillin1/ HSP70 non-EV: Grp78/ GRP78/ COXIV/ Parp Proteomics yes Show all info Study aim Function/Identification of content (omics approaches) Sample Species Rattus norvegicus Sample Type Cell culture supernatant EV-producing cells Primary rat hepatocytes EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g Cell count 300000000 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type Type 45 Ti Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 45 Wash: time (min) 90 Wash: Rotor Type Type 45 Ti Wash: speed (g) 100000 Density gradient Only used for validation of main results Yes Type Discontinuous Number of initial discontinuous layers 2 Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 16,5 Sample volume (mL) 5,5 Orientation Top-down Speed (g) 100000 Duration (min) 160 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction Not repo Pelleting: duration (min) 60 Pelleting: rotor type Not reported Pelleting: speed (g) 100000 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method Bradford Western Blot Detected EV-associated proteins Flotillin1/ CD63/ HSP90/ TSG101/ HSP70/ Alix/ CD81 Not detected EV-associated proteins HSP90/ HSP70/ CD81/ Flotillin1/ TSG101/ CD63/ Alix Detected contaminants COXIV/ GRP78/ Parp Not detected contaminants COXIV/ Grp78/ Parp Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 50-400 EV concentration Yes Particle yield as number of particles per million cells: 1,8 EM EM-type Cryo-EM Image type Close-up

	EV200159	2/4	Homo sapiens	Expi293F	DG (d)(U)C	Lázaro-Ibáñez, Elisa	2021	89%
Study summary Full title Selection of Fluorescent, Bioluminescent, and Radioactive Tracers to Accurately Reflect Extracellular Vesicle Biodistribution in Vivo All authors Elisa Lázaro-Ibáñez, Farid N Faruqu, Amer F Saleh, Andreia M Silva, Julie Tzu-Wen Wang, Janusz Rak, Khuloud T Al-Jamal, Niek Dekker Journal ACS Nano Abstract The ability to track extracellular vesicles (EVs) in vivo without influencing their biodistribution (show more...)The ability to track extracellular vesicles (EVs) in vivo without influencing their biodistribution is a key requirement for their successful development as drug delivery vehicles and therapeutic agents. Here, we evaluated the effect of five different optical and nuclear tracers on the in vivo biodistribution of EVs. Expi293F EVs were labeled using either a noncovalent fluorescent dye DiR, or covalent modification with 111indium-DTPA, or bioengineered with fluorescent (mCherry) or bioluminescent (Firefly and NanoLuc luciferase) proteins fused to the EV marker, CD63. To focus specifically on the effect of the tracer, we compared EVs derived from the same cell source and administered systemically by the same route and at equal dose into tumor-bearing BALB/c mice. 111Indium and DiR were the most sensitive tracers for in vivo imaging of EVs, providing the most accurate quantification of vesicle biodistribution by ex vivo imaging of organs and analysis of tissue lysates. Specifically, NanoLuc fused to CD63 altered EV distribution, resulting in high accumulation in the lungs, demonstrating that genetic modification of EVs for tracking purposes may compromise their physiological biodistribution. Blood kinetic analysis revealed that EVs are rapidly cleared from the circulation with a half-life below 10 min. Our study demonstrates that radioactivity is the most accurate EV tracking approach for a complete quantitative biodistribution study including pharmacokinetic profiling. In conclusion, we provide a comprehensive comparison of fluorescent, bioluminescent, and radioactivity approaches, including dual labeling of EVs, to enable accurate spatiotemporal resolution of EV trafficking in mice, an essential step in developing EV therapeutics. (hide) EV-METRIC 89% (99th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin CD63-mCherry Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture Density gradient (Differential) (ultra)centrifugation Protein markers EV: CD63/ CD81/ Alix/ Flotillin1/ CD9/ mcherry non-EV: Lamin B1 Proteomics no EV density (g/ml) 1.10 - 1.13 Show all info Study aim Technical analysis comparing/optimizing EV-related methods/Mechanism of uptake/transfer Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Expi293F EV-harvesting Medium Serum free medium Cell viability (%) 85 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type Type 45 Ti Pelleting: speed (g) 100000 Density gradient Type Discontinuous Number of initial discontinuous layers 9 Lowest density fraction 10% Highest density fraction 50% Total gradient volume, incl. sample (mL) 17 Sample volume (mL) 1 Orientation Bottom-up Rotor type SW 32.1 Ti Speed (g) 120000 Duration (min) 960 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 94 Pelleting: duration (min) 180 Pelleting: rotor type Type 45 Ti Pelleting: speed (g) 120000 Characterization: Protein analysis Protein Concentration Method Fluorometric assay (e.g. Qubit, NanoOrange,...) Western Blot Detected EV-associated proteins Flotillin1/ CD9/ CD63/ mCherry/ Alix/ CD81 Not detected contaminants Lamin B1 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 126-154 EV concentration Yes Particle yield particles per milliliter of final volume of sample;Yes, other: 5,00E+13 EM EM-type Transmission-EM Image type Close-up, Wide-field Report size (nm) 56

	EV200159	3/4	Homo sapiens	Expi293F	DG (d)(U)C	Lázaro-Ibáñez, Elisa	2021	89%
Study summary Full title Selection of Fluorescent, Bioluminescent, and Radioactive Tracers to Accurately Reflect Extracellular Vesicle Biodistribution in Vivo All authors Elisa Lázaro-Ibáñez, Farid N Faruqu, Amer F Saleh, Andreia M Silva, Julie Tzu-Wen Wang, Janusz Rak, Khuloud T Al-Jamal, Niek Dekker Journal ACS Nano Abstract The ability to track extracellular vesicles (EVs) in vivo without influencing their biodistribution (show more...)The ability to track extracellular vesicles (EVs) in vivo without influencing their biodistribution is a key requirement for their successful development as drug delivery vehicles and therapeutic agents. Here, we evaluated the effect of five different optical and nuclear tracers on the in vivo biodistribution of EVs. Expi293F EVs were labeled using either a noncovalent fluorescent dye DiR, or covalent modification with 111indium-DTPA, or bioengineered with fluorescent (mCherry) or bioluminescent (Firefly and NanoLuc luciferase) proteins fused to the EV marker, CD63. To focus specifically on the effect of the tracer, we compared EVs derived from the same cell source and administered systemically by the same route and at equal dose into tumor-bearing BALB/c mice. 111Indium and DiR were the most sensitive tracers for in vivo imaging of EVs, providing the most accurate quantification of vesicle biodistribution by ex vivo imaging of organs and analysis of tissue lysates. Specifically, NanoLuc fused to CD63 altered EV distribution, resulting in high accumulation in the lungs, demonstrating that genetic modification of EVs for tracking purposes may compromise their physiological biodistribution. Blood kinetic analysis revealed that EVs are rapidly cleared from the circulation with a half-life below 10 min. Our study demonstrates that radioactivity is the most accurate EV tracking approach for a complete quantitative biodistribution study including pharmacokinetic profiling. In conclusion, we provide a comprehensive comparison of fluorescent, bioluminescent, and radioactivity approaches, including dual labeling of EVs, to enable accurate spatiotemporal resolution of EV trafficking in mice, an essential step in developing EV therapeutics. (hide) EV-METRIC 89% (99th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin CD63-FLuc Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture Density gradient (Differential) (ultra)centrifugation Protein markers EV: CD63/ CD81/ Alix/ Flotillin1/ CD9 non-EV: Lamin B1 Proteomics no EV density (g/ml) 1.10 - 1.13 Show all info Study aim Technical analysis comparing/optimizing EV-related methods/Mechanism of uptake/transfer Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Expi293F EV-harvesting Medium Serum free medium Cell viability (%) 85 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type Type 45 Ti Pelleting: speed (g) 100000 Density gradient Type Discontinuous Number of initial discontinuous layers 9 Lowest density fraction 10% Highest density fraction 50% Total gradient volume, incl. sample (mL) 17 Sample volume (mL) 1 Orientation Bottom-up Rotor type SW 32.1 Ti Speed (g) 120000 Duration (min) 960 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 94 Pelleting: duration (min) 180 Pelleting: rotor type Type 45 Ti Pelleting: speed (g) 120000 Characterization: Protein analysis Protein Concentration Method Fluorometric assay (e.g. Qubit, NanoOrange,...) Western Blot Detected EV-associated proteins Flotillin1/ Alix/ Firely luciferase/ CD9/ CD63/ CD81 Not detected contaminants Lamin B1 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 126-154 EV concentration Yes Particle yield particles per milliliter of final volume of sample;Yes, other: 1,50E+13 EM EM-type Transmission-EM Image type Close-up, Wide-field Report size (nm) 80

	EV200157	5/10	Homo sapiens	MDA-MB-468	(d)(U)C SEC (non-commercial) Polymer-based precipitation DG	Martínez-Greene, Juan A	2021	89%
Study summary Full title Quantitative proteomic analysis of extracellular vesicle subgroups isolated by an optimized method combining polymer-based precipitation and size exclusion chromatography All authors Juan A Martínez-Greene, Karina Hernández-Ortega, Ricardo Quiroz-Baez, Osbaldo Resendis-Antonio, Israel Pichardo-Casas, David A Sinclair, Bogdan Budnik, Alfredo Hidalgo-Miranda, Eileen Uribe-Querol, María Del Pilar Ramos-Godínez, Eduardo Martínez-Martínez Journal J Extracell Vesicles Abstract The molecular characterization of extracellular vesicles (EVs) has revealed a great heterogeneity in (show more...)The molecular characterization of extracellular vesicles (EVs) has revealed a great heterogeneity in their composition at a cellular and tissue level. Current isolation methods fail to efficiently separate EV subtypes for proteomic and functional analysis. The aim of this study was to develop a reproducible and scalable isolation workflow to increase the yield and purity of EV preparations. Through a combination of polymer-based precipitation and size exclusion chromatography (Pre-SEC), we analyzed two subsets of EVs based on their CD9, CD63 and CD81 content and elution time. EVs were characterized using transmission electron microscopy, nanoparticle tracking analysis, and Western blot assays. To evaluate differences in protein composition between the early- and late-eluting EV fractions, we performed a quantitative proteomic analysis of MDA-MB-468-derived EVs. We identified 286 exclusive proteins in early-eluting fractions and 148 proteins with a differential concentration between early- and late-eluting fractions. A density gradient analysis further revealed EV heterogeneity within each analyzed subgroup. Through a systems biology approach, we found significant interactions among proteins contained in the EVs which suggest the existence of functional clusters related to specific biological processes. The workflow presented here allows the study of EV subtypes within a single cell type and contributes to standardizing the EV isolation for functional studies. (hide) EV-METRIC 89% (99th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Size-exclusion chromatography (non-commercial) Polymer-based precipitation Density gradient Protein markers EV: CD9/ CD63/ CD81/ Alix/ TSG101/ ANXA2/ ANXA5 non-EV: Albumin Proteomics yes EV density (g/ml) 1.08-1.15 Show all info Study aim New methodological development/Biomarker/Identification of content (omics approaches)/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells MDA-MB-468 EV-harvesting Medium EV-depleted medium Preparation of EDS >=18h at >= 100,000g Cell viability (%) 95 Cell count 1.50E+06 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 39 Pelleting: rotor type TLA-100.3 Pelleting: speed (g) 118000 Density gradient Type Discontinuous Number of initial discontinuous layers 3 Lowest density fraction 10% Highest density fraction 30% Total gradient volume, incl. sample (mL) 5 Sample volume (mL) 2.5 Orientation Bottom-up Rotor type SW 55 Ti Speed (g) 200000 Duration (min) 60 Fraction volume (mL) 0.49 Fraction processing Centrifugation Pelleting: volume per fraction 2.8 Pelleting: duration (min) 39 Pelleting: rotor type TLA-100.3 Pelleting: speed (g) 118000 Size-exclusion chromatography Total column volume (mL) 10 Sample volume/column (mL) 1 Resin type Sepharose CL-2B Other Name other separation method Polymer-based precipitation EV-subtype Distinction between multiple subtypes SEC fraction Used subtypes F5-10 Characterization: Protein analysis Protein Concentration Method BCA Western Blot Detected EV-associated proteins CD9/ CD63/ CD81/ Alix/ TSG101/ ANXA2/ ANXA5 Detected contaminants Albumin Proteomics database Yes: ProteomeXchange Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 148.9 EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 4.13E+11 EM EM-type Transmission-EM Image type Wide-field

	EV200157	6/10	Homo sapiens	MDA-MB-468	(d)(U)C SEC (non-commercial) Polymer-based precipitation DG	Martínez-Greene, Juan A	2021	89%
Study summary Full title Quantitative proteomic analysis of extracellular vesicle subgroups isolated by an optimized method combining polymer-based precipitation and size exclusion chromatography All authors Juan A Martínez-Greene, Karina Hernández-Ortega, Ricardo Quiroz-Baez, Osbaldo Resendis-Antonio, Israel Pichardo-Casas, David A Sinclair, Bogdan Budnik, Alfredo Hidalgo-Miranda, Eileen Uribe-Querol, María Del Pilar Ramos-Godínez, Eduardo Martínez-Martínez Journal J Extracell Vesicles Abstract The molecular characterization of extracellular vesicles (EVs) has revealed a great heterogeneity in (show more...)The molecular characterization of extracellular vesicles (EVs) has revealed a great heterogeneity in their composition at a cellular and tissue level. Current isolation methods fail to efficiently separate EV subtypes for proteomic and functional analysis. The aim of this study was to develop a reproducible and scalable isolation workflow to increase the yield and purity of EV preparations. Through a combination of polymer-based precipitation and size exclusion chromatography (Pre-SEC), we analyzed two subsets of EVs based on their CD9, CD63 and CD81 content and elution time. EVs were characterized using transmission electron microscopy, nanoparticle tracking analysis, and Western blot assays. To evaluate differences in protein composition between the early- and late-eluting EV fractions, we performed a quantitative proteomic analysis of MDA-MB-468-derived EVs. We identified 286 exclusive proteins in early-eluting fractions and 148 proteins with a differential concentration between early- and late-eluting fractions. A density gradient analysis further revealed EV heterogeneity within each analyzed subgroup. Through a systems biology approach, we found significant interactions among proteins contained in the EVs which suggest the existence of functional clusters related to specific biological processes. The workflow presented here allows the study of EV subtypes within a single cell type and contributes to standardizing the EV isolation for functional studies. (hide) EV-METRIC 89% (99th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Size-exclusion chromatography (non-commercial) Polymer-based precipitation Density gradient Protein markers EV: CD9/ CD63/ CD81/ Alix/ TSG101/ ANXA2/ ANXA5 non-EV: Albumin Proteomics yes EV density (g/ml) 1.08-1.15 Show all info Study aim New methodological development/Biomarker/Identification of content (omics approaches)/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells MDA-MB-468 EV-harvesting Medium EV-depleted medium Preparation of EDS >=18h at >= 100,000g Cell viability (%) 95 Cell count 1.50E+06 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 39 Pelleting: rotor type TLA-100.3 Pelleting: speed (g) 118000 Density gradient Type Discontinuous Number of initial discontinuous layers 3 Lowest density fraction 10% Highest density fraction 30% Total gradient volume, incl. sample (mL) 5 Sample volume (mL) 2.5 Orientation Bottom-up Rotor type SW 55 Ti Speed (g) 200000 Duration (min) 60 Fraction volume (mL) 0.49 Fraction processing Centrifugation Pelleting: volume per fraction 2.8 Pelleting: duration (min) 39 Pelleting: rotor type TLA-100.3 Pelleting: speed (g) 118000 Size-exclusion chromatography Total column volume (mL) 10 Sample volume/column (mL) 1 Resin type Sepharose CL-2B Other Name other separation method Polymer-based precipitation EV-subtype Distinction between multiple subtypes SEC fraction Used subtypes F11-16 Characterization: Protein analysis Western Blot Detected EV-associated proteins CD9/ CD63/ CD81/ ANXA2/ ANXA5 Not detected EV-associated proteins Alix/ TSG101 Detected contaminants Albumin Proteomics database Yes: ProteomeXchange Characterization: Particle analysis NTA Report type Mean Reported size (nm) 124 EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 4.29E+11 EM EM-type Transmission-EM Image type Wide-field

	EV210179	2/6	Mus musculus	Renca	(d)(U)C DG	Samoylenko, Anatoliy	2021	88%
Study summary Full title Time-gated Raman spectroscopy and proteomics analyses of hypoxic and normoxic renal carcinoma extracellular vesicles All authors Anatoliy Samoylenko, Martin Kögler, Artem Zhyvolozhnyi, Olha Makieieva, Geneviève Bart, Sampson S. Andoh, Matthieu Roussey, Seppo J. Vainio, and Jussi Hiltunen Journal Sci Rep Abstract Extracellular vesicles (EVs) represent a diverse group of small membrane-encapsulated particles invo (show more...)Extracellular vesicles (EVs) represent a diverse group of small membrane-encapsulated particles involved in cell–cell communication, but the technologies to characterize EVs are still limited. Hypoxia is a typical condition in solid tumors, and cancer-derived EVs support tumor growth and invasion of tissues by tumor cells. We found that exposure of renal adenocarcinoma cells to hypoxia induced EV secretion and led to notable changes in the EV protein cargo in comparison to normoxia. Proteomics analysis showed overrepresentation of proteins involved in adhesion, such as integrins, in hypoxic EV samples. We further assessed the efficacy of time-gated Raman spectroscopy (TG-RS) and surface-enhanced time-gated Raman spectroscopy (TG-SERS) to characterize EVs. While the conventional continuous wave excitation Raman spectroscopy did not provide a notable signal, prominent signals were obtained with the TG-RS that were further enhanced in the TG-SERS. The Raman signal showed characteristic changes in the amide regions due to alteration in the chemical bonds of the EV proteins. The results illustrate that the TG-RS and the TG-SERS are promising label free technologies to study cellular impact of external stimuli, such as oxygen deficiency, on EV production, as well as differences arising from distinct EV purification protocols. (hide) EV-METRIC 88% (98th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C DG Protein markers EV: TSG101/ Alix/ CD9/ CD81 non-EV: Argonaute2/ GM130 Proteomics no EV density (g/ml) 1.07-1.12 Show all info Study aim New methodological development/Identification of content (omics approaches) Sample Species Mus musculus Sample Type Cell culture supernatant EV-producing cells Renca EV-harvesting Medium Serum free medium Cell viability (%) 97 Cell count Not reported Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Pelleting performed No Density gradient Type Continuous Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 12 Sample volume (mL) 2 Orientation Top-down Rotor type TH-641 Speed (g) 100000 Duration (min) 900 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 10 Pelleting: duration (min) 900 Pelleting: rotor type TH-641 Pelleting: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method BCA Protein Yield (µg) Yes, per cell 0.17 Western Blot Detected EV-associated proteins CD9/ TSG101/ Alix/ CD81 Not detected contaminants GM130/ Argonaute2 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 108 EV concentration Yes Particle yield Yes, as number of particles per million cells 9.96E+06 EM EM-type Immuno-EM/ Transmission-EM EM protein CD63 Image type Wide-field Report size (nm) 30-200

	EV210179	4/6	Mus musculus	Renca	(d)(U)C DG	Samoylenko, Anatoliy	2021	88%
Study summary Full title Time-gated Raman spectroscopy and proteomics analyses of hypoxic and normoxic renal carcinoma extracellular vesicles All authors Anatoliy Samoylenko, Martin Kögler, Artem Zhyvolozhnyi, Olha Makieieva, Geneviève Bart, Sampson S. Andoh, Matthieu Roussey, Seppo J. Vainio, and Jussi Hiltunen Journal Sci Rep Abstract Extracellular vesicles (EVs) represent a diverse group of small membrane-encapsulated particles invo (show more...)Extracellular vesicles (EVs) represent a diverse group of small membrane-encapsulated particles involved in cell–cell communication, but the technologies to characterize EVs are still limited. Hypoxia is a typical condition in solid tumors, and cancer-derived EVs support tumor growth and invasion of tissues by tumor cells. We found that exposure of renal adenocarcinoma cells to hypoxia induced EV secretion and led to notable changes in the EV protein cargo in comparison to normoxia. Proteomics analysis showed overrepresentation of proteins involved in adhesion, such as integrins, in hypoxic EV samples. We further assessed the efficacy of time-gated Raman spectroscopy (TG-RS) and surface-enhanced time-gated Raman spectroscopy (TG-SERS) to characterize EVs. While the conventional continuous wave excitation Raman spectroscopy did not provide a notable signal, prominent signals were obtained with the TG-RS that were further enhanced in the TG-SERS. The Raman signal showed characteristic changes in the amide regions due to alteration in the chemical bonds of the EV proteins. The results illustrate that the TG-RS and the TG-SERS are promising label free technologies to study cellular impact of external stimuli, such as oxygen deficiency, on EV production, as well as differences arising from distinct EV purification protocols. (hide) EV-METRIC 88% (98th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin hypoxia Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C DG Protein markers EV: Alix/ TSG101/ CD9/ CD81 non-EV: Argonaute2/ GM130 Proteomics no EV density (g/ml) 1.07-1.12 Show all info Study aim New methodological development/Identification of content (omics approaches) Sample Species Mus musculus Sample Type Cell culture supernatant EV-producing cells Renca EV-harvesting Medium Serum free medium Cell viability (%) 97 Cell count Not reported Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Pelleting performed No Density gradient Type Continuous Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 12 Sample volume (mL) 2 Orientation Top-down Rotor type TH-641 Speed (g) 100000 Duration (min) 900 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 10 Pelleting: duration (min) 900 Pelleting: rotor type TH-641 Pelleting: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method BCA Protein Yield (µg) Yes, per cell 0.185 Western Blot Detected EV-associated proteins Alix/ CD9/ TSG101/ CD81 Not detected contaminants GM130/ Argonaute2 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 128 EV concentration Yes Particle yield Yes, as number of particles per million cells 2.10E+07 EM EM-type Immuno-EM/ Transmission-EM EM protein CD63 Image type Wide-field Report size (nm) 30-200

	EV200081	1/4	Homo sapiens	Blood plasma	DG SEC (non-commercial)	Vergauwen, Glenn	2021	88%
Study summary Full title Robust sequential biophysical fractionation of blood plasma to study variations in the biomolecular landscape of systemically circulating extracellular vesicles across clinical conditions All authors Glenn Vergauwen, Joeri Tulkens, Cláudio Pinheiro, Francisco Avila Cobos, Sándor Dedeyne, Marie-Angélique De Scheerder, Linos Vandekerckhove, Francis Impens, Ilkka Miinalainen, Geert Braems, Kris Gevaert, Pieter Mestdagh, Jo Vandesompele, Hannelore Denys, Olivier De Wever, An Hendrix Journal J Extracell Vesicles Abstract Separating extracellular vesicles (EV) from blood plasma is challenging and complicates their biolog (show more...)Separating extracellular vesicles (EV) from blood plasma is challenging and complicates their biological understanding and biomarker development. In this study, we fractionate blood plasma by combining size-exclusion chromatography (SEC) and OptiPrep density gradient centrifugation to study clinical context-dependent and time-dependent variations in the biomolecular landscape of systemically circulating EV. Using pooled blood plasma samples from breast cancer patients, we first demonstrate the technical repeatability of blood plasma fractionation. Using serial blood plasma samples from HIV and ovarian cancer patients (n = 10) we next show that EV carry a clinical context-dependent and/or time-dependent protein and small RNA composition, including miRNA and tRNA. In addition, differential analysis of blood plasma fractions provides a catalogue of putative proteins not associated with systemically circulating EV. In conclusion, the implementation of blood plasma fractionation allows to advance the biological understanding and biomarker development of systemically circulating EV. (hide) EV-METRIC 88% (99th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Blood plasma Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture Density gradient Size-exclusion chromatography (non-commercial) Protein markers EV: Flotillin1/ CD9 non-EV: APOB/ APOA1 Proteomics no EV density (g/ml) 1.09-1.10 Show all info Study aim Biomarker/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Blood plasma Separation Method Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 16.5 Sample volume (mL) 1 Orientation Top-down Rotor type SW 32.1 Ti Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 1 Fraction processing Size-exclusion chromatography Size-exclusion chromatography Total column volume (mL) 10 Sample volume/column (mL) 2 Resin type Sepharose CL-2B Characterization: Protein analysis Protein Concentration Method Fluorometric assay (e.g. Qubit, NanoOrange,...) Western Blot Detected EV-associated proteins Flotillin1 Not detected contaminants APOA1 ELISA Detected EV-associated proteins CD9 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Not Reported EM EM-type Transmission-EM Image type Close-up, Wide-field Report size (nm) 50-250

	EV200102	4/7	Homo sapiens	Blood plasma	DG	Tóth, Eszter	2021	86%
Study summary Full title Formation of a protein corona on the surface of extracellular vesicles in blood plasma All authors Eszter Á Tóth, Lilla Turiák, Tamás Visnovitz, Csaba Cserép, Anett Mázló, Barbara W Sódar, András I Försönits, Gábor Petővári, Anna Sebestyén, Zsolt Komlósi, László Drahos, Ágnes Kittel, György Nagy, Attila Bácsi, Ádám Dénes, Yong Song Gho, Katalin É Szabó-Taylor, Edit I Buzás Journal J Extracell Vesicles Abstract In this study we tested whether a protein corona is formed around extracellular vesicles (EVs) in bl (show more...)In this study we tested whether a protein corona is formed around extracellular vesicles (EVs) in blood plasma. We isolated medium-sized nascent EVs of THP1 cells as well as of Optiprep-purified platelets, and incubated them in EV-depleted blood plasma from healthy subjects and from patients with rheumatoid arthritis. EVs were subjected to differential centrifugation, size exclusion chromatography, or density gradient ultracentrifugation followed by mass spectrometry. Plasma protein-coated EVs had a higher density compared to the nascent ones and carried numerous newly associated proteins. Interactions between plasma proteins and EVs were confirmed by confocal microscopy, capillary Western immunoassay, immune electron microscopy and flow cytometry. We identified nine shared EV corona proteins (ApoA1, ApoB, ApoC3, ApoE, complement factors 3 and 4B, fibrinogen α-chain, immunoglobulin heavy constant γ2 and γ4 chains), which appear to be common corona proteins among EVs, viruses and artificial nanoparticles in blood plasma. An unexpected finding of this study was the high overlap of the composition of the protein corona with blood plasma protein aggregates. This is explained by our finding that besides a diffuse, patchy protein corona, large protein aggregates also associate with the surface of EVs. However, while EVs with an external plasma protein cargo induced an increased expression of TNF-α, IL-6, CD83, CD86 and HLA-DR of human monocyte-derived dendritic cells, EV-free protein aggregates had no effect. In conclusion, our data may shed new light on the origin of the commonly reported plasma protein 'contamination' of EV preparations and may add a new perspective to EV research. (hide) EV-METRIC 86% (98th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Blood plasma Sample origin Control: EV-depleted plasma, spiked with THP1 EVs Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture Density gradient Protein markers EV: CD63/ Phosphatydilserine non-EV: None Proteomics yes EV density (g/ml) 1.10-1.15 Show all info Study aim Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Blood plasma Separation Method Density gradient Only used for validation of main results Yes Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 4.5 Sample volume (mL) 0.5 Orientation Top-down Rotor type MLS-50 Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 0.5 Fraction processing Centrifugation Pelleting: volume per fraction 2 Pelleting: duration (min) 80 Pelleting: rotor type FA-45-24-11 Pelleting: speed (g) 12500 Characterization: Protein analysis Protein Concentration Method microBCA Flow cytometry Type of Flow cytometry FACS Calibur Calibration bead size The vesicular gate was set using Megamix Beads (Bi Detected EV-associated proteins Phosphatydilserine Proteomics database Yes: Characterization: Lipid analysis No Characterization: Particle analysis Particle analysis: flow cytometry Flow cytometer type FACS Calibur Hardware adjustment Calibration bead size 0.160;0.200;0.240;0.500 Report type Not Reported EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV210209	6/7	Homo sapiens	VERO-E6	(d)(U)C DG	Xie F	2021	78%
Study summary Full title Engineering Extracellular Vesicles Enriched with Palmitoylated ACE2 as COVID-19 Therapy. All authors Xie F, Su P, Pan T, Zhou X, Li H, Huang H, Wang A, Wang F, Huang J, Yan H, Zeng L, Zhang L, Zhou F Journal Adv Mater Abstract Angiotensin converting enzyme 2 (ACE2) is a key receptor present on cell surfaces that directly inte (show more...)Angiotensin converting enzyme 2 (ACE2) is a key receptor present on cell surfaces that directly interacts with the viral spike (S) protein of the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). It is proposed that inhibiting this interaction can be promising in treating COVID-19. Here, the presence of ACE2 in extracellular vesicles (EVs) is reported and the EV-ACE2 levels are determined by protein palmitoylation. The Cys141 and Cys498 residues on ACE2 are S-palmitoylated by zinc finger DHHC-Type Palmitoyltransferase 3 (ZDHHC3) and de-palmitoylated by acyl protein thioesterase 1 (LYPLA1), which is critical for the membrane-targeting of ACE2 and their EV secretion. Importantly, by fusing the S-palmitoylation-dependent plasma membrane (PM) targeting sequence with ACE2, EVs enriched with ACE2 on their surface (referred to as PM-ACE2-EVs) are engineered. It is shown that PM-ACE2-EVs can bind to the SARS-CoV-2 S-RBD with high affinity and block its interaction with cell surface ACE2 in vitro. PM-ACE2-EVs show neutralization potency against pseudotyped and authentic SARS-CoV-2 in human ACE2 (hACE2) transgenic mice, efficiently block viral load of authentic SARS-CoV-2, and thus protect host against SARS-CoV-2-induced lung inflammation. The study provides an efficient engineering protocol for constructing a promising, novel biomaterial for application in prophylactic and therapeutic treatments against COVID-19. (hide) EV-METRIC 78% (97th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles exosome Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Density gradient Protein markers EV: Alix/ TSG101/ ACE2 non-EV: None Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells VERO-E6 EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 120000 Wash: volume per pellet (ml) 36mL/ultracentrifuge tube Wash: time (min) 70 Wash: Rotor Type SW 32 Ti Wash: speed (g) 120000 Density gradient Only used for validation of main results Yes Type Discontinuous Number of initial discontinuous layers 5 Lowest density fraction 5% Highest density fraction 60% Orientation Top-down Speed (g) 100,000g Duration (min) 1080 Other Name other separation method Density gradient EV-subtype Distinction between multiple subtypes Size Used subtypes 30-200 nm Characterization: Protein analysis Protein Concentration Method BCA Western Blot Detected EV-associated proteins TSG101/ Alix/ ACE2 ELISA Detected EV-associated proteins ACE2 Fluorescent NTA Antibody details provided? No Detected EV-associated proteins ACE2-GFP Characterization: Lipid analysis No Characterization: Particle analysis DLS Report type Size range/distribution Used for determining EV concentration? Yes EM EM-type Transmission-EM/ Immuno-EM EM protein ACE2/ TSG101 Image type Close-up

	EV210180	1/2	Homo sapiens	SW620	(d)(U)C	Santos, Mark F	2021	78%
Study summary Full title Itraconazole inhibits nuclear delivery of extracellular vesicle cargo by disrupting the entry of late endosomes into the nucleoplasmic reticulum All authors Mark F. Santos, Germana Rappa, Jana Karbanová, Simona Fontana, Maria Antonietta Di Bella, Marshall R. Pope, Barbara Parrino, Stella Maria Cascioferro, Giulio Vistoli, Patrizia Diana, Girolamo Cirrincione, Goffredo O. Arena, Gyunghwi Woo, Kevin Huang, Tony Huynh, Marta Moschetti, Riccardo Alessandro, Denis Corbeil, Aurelio Lorico Journal J Extracell Vesicles Abstract Extracellular vesicles (EVs) are mediators of intercellular communication under both healthy and pat (show more...)Extracellular vesicles (EVs) are mediators of intercellular communication under both healthy and pathological conditions, including the induction of pro-metastatic traits, but it is not yet known how and where functional cargoes of EVs are delivered to their targets in host cell compartments. We have described that after endocytosis, EVs reach Rab7+ late endosomes and a fraction of these enter the nucleoplasmic reticulum and transport EV biomaterials to the host cell nucleoplasm. Their entry therein and docking to outer nuclear membrane occur through a tripartite complex formed by the proteins VAP-A, ORP3 and Rab7 (VOR complex). Here, we report that the antifungal compound itraconazole (ICZ), but not its main metabolite hydroxy-ICZ or ketoconazole, disrupts the binding of Rab7 to ORP3–VAP-A complexes, leading to inhibition of EV-mediated pro-metastatic morphological changes including cell migration behaviour of colon cancer cells. With novel, smaller chemical drugs, inhibition of the VOR complex was maintained, although the ICZ moieties responsible for antifungal activity and interference with intracellular cholesterol distribution were removed. Knowing that cancer cells hijack their microenvironment and that EVs derived from them determine the pre-metastatic niche, small-sized inhibitors of nuclear transfer of EV cargo into host cells could find cancer therapeutic applications, particularly in combination with direct targeting of cancer cells. (hide) EV-METRIC 78% (97th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Protein markers EV: CD63/ CD81/ Alix/ CD9 non-EV: Histone H1/ Calnexin Proteomics no Show all info Study aim Function/Mechanism of uptake/transfer Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells SW620 EV-harvesting Medium Serum free medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Equal to or above 150,000 g Pelleting performed Yes Pelleting: time(min) 60 Pelleting: rotor type SW 55 Ti Pelleting: speed (g) 200000 Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Detected EV-associated proteins CD9/ CD63/ Alix/ CD81 Not detected contaminants Histone H1/ Calnexin Detected EV-associated proteins CD9/ CD63/ CD81 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 150 EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 4.00E+10 EM EM-type Transmission-EM Image type Close-up, Wide-field Report size (nm) 60-130

	EV210180	2/2	Homo sapiens	FEMX-I	(d)(U)C	Santos, Mark F	2021	78%
Study summary Full title Itraconazole inhibits nuclear delivery of extracellular vesicle cargo by disrupting the entry of late endosomes into the nucleoplasmic reticulum All authors Mark F. Santos, Germana Rappa, Jana Karbanová, Simona Fontana, Maria Antonietta Di Bella, Marshall R. Pope, Barbara Parrino, Stella Maria Cascioferro, Giulio Vistoli, Patrizia Diana, Girolamo Cirrincione, Goffredo O. Arena, Gyunghwi Woo, Kevin Huang, Tony Huynh, Marta Moschetti, Riccardo Alessandro, Denis Corbeil, Aurelio Lorico Journal J Extracell Vesicles Abstract Extracellular vesicles (EVs) are mediators of intercellular communication under both healthy and pat (show more...)Extracellular vesicles (EVs) are mediators of intercellular communication under both healthy and pathological conditions, including the induction of pro-metastatic traits, but it is not yet known how and where functional cargoes of EVs are delivered to their targets in host cell compartments. We have described that after endocytosis, EVs reach Rab7+ late endosomes and a fraction of these enter the nucleoplasmic reticulum and transport EV biomaterials to the host cell nucleoplasm. Their entry therein and docking to outer nuclear membrane occur through a tripartite complex formed by the proteins VAP-A, ORP3 and Rab7 (VOR complex). Here, we report that the antifungal compound itraconazole (ICZ), but not its main metabolite hydroxy-ICZ or ketoconazole, disrupts the binding of Rab7 to ORP3–VAP-A complexes, leading to inhibition of EV-mediated pro-metastatic morphological changes including cell migration behaviour of colon cancer cells. With novel, smaller chemical drugs, inhibition of the VOR complex was maintained, although the ICZ moieties responsible for antifungal activity and interference with intracellular cholesterol distribution were removed. Knowing that cancer cells hijack their microenvironment and that EVs derived from them determine the pre-metastatic niche, small-sized inhibitors of nuclear transfer of EV cargo into host cells could find cancer therapeutic applications, particularly in combination with direct targeting of cancer cells. (hide) EV-METRIC 78% (97th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin CD9-GFP expression Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Protein markers EV: CD63/ CD81/ Alix/ CD9 non-EV: Histone H1/ Calnexin Proteomics no Show all info Study aim Function/Mechanism of uptake/transfer Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells FEMX-I EV-harvesting Medium Serum free medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Equal to or above 150,000 g Pelleting performed Yes Pelleting: time(min) 60 Pelleting: rotor type SW 55 Ti Pelleting: speed (g) 200000 Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Detected EV-associated proteins CD9/ CD63/ Alix/ CD81 Not detected contaminants Histone H1/ Calnexin Detected EV-associated proteins CD9/ CD63 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 120 EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 2.00E+10 EM EM-type Transmission-EM Image type Close-up, Wide-field Report size (nm) 40-100

	EV210166	5/6	Homo sapiens	Blood plasma	(d)(U)C	Krishnamachary, Balaji	2021	78%
Study summary Full title Extracellular vesicle-mediated endothelial apoptosis and EV-associated proteins correlate with COVID-19 disease severity All authors Balaji Krishnamachary, Christine Cook, Ashok Kumar, Leslie Spikes, Prabhakar Chalise, Navneet K Dhillon Journal J Extracell Vesicles Abstract Coronavirus disease-2019 (COVID-19), caused by the novel severe acute respiratory syndrome coronavir (show more...)Coronavirus disease-2019 (COVID-19), caused by the novel severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), has lead to a global pandemic with a rising toll in infections and deaths. Better understanding of its pathogenesis will greatly improve the outcomes and treatment of affected patients. Here we compared the inflammatory and cardiovascular disease-related protein cargo of circulating large and small extracellular vesicles (EVs) from 84 hospitalized patients infected with SARS-CoV-2 with different stages of disease severity. Our findings reveal significant enrichment of proinflammatory, procoagulation, immunoregulatory and tissue-remodelling protein signatures in EVs, which remarkably distinguished symptomatic COVID-19 patients from uninfected controls with matched comorbidities and delineated those with moderate disease from those who were critically ill. Specifically, EN-RAGE, followed by TF and IL-18R1, showed the strongest correlation with disease severity and length of hospitalization. Importantly, EVs from COVID-19 patients induced apoptosis of pulmonary microvascular endothelial cells in the order of disease severity. In conclusion, our findings support a role for EVs in the pathogenesis of COVID-19 disease and underpin the development of EV-based approaches to predicting disease severity, determining need for patient hospitalization and identifying new therapeutic targets. (hide) EV-METRIC 78% (98th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Blood plasma Sample origin COVID19 Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Protein markers EV: TSG101/ Alix/ CD63/ CD81/ Integrin beta1/ Flotillin1/ CD9 non-EV: ApoE/ GM130 Proteomics no Show all info Study aim Function/Biomarker Sample Species Homo sapiens Sample Type Blood plasma Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 15 Pelleting: rotor type FA-45-24-11 Pelleting: speed (g) 20000 Wash: volume per pellet (ml) 1 Wash: time (min) 15 Wash: Rotor Type FA-45-24-11 Wash: speed (g) 20000 Characterization: Protein analysis Protein Concentration Method BCA Western Blot Detected EV-associated proteins Flotillin1/ Integrin beta1/ CD9/ CD63/ TSG101/ Alix/ CD81 Detected contaminants ApoE Not detected contaminants GM130 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 50-300 EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 7.10E+10 EM EM-type Transmission-EM Image type Close-up, Wide-field Report size (nm) 100-450

	EV210166	6/6	Homo sapiens	Blood plasma	(d)(U)C	Krishnamachary, Balaji	2021	78%
Study summary Full title Extracellular vesicle-mediated endothelial apoptosis and EV-associated proteins correlate with COVID-19 disease severity All authors Balaji Krishnamachary, Christine Cook, Ashok Kumar, Leslie Spikes, Prabhakar Chalise, Navneet K Dhillon Journal J Extracell Vesicles Abstract Coronavirus disease-2019 (COVID-19), caused by the novel severe acute respiratory syndrome coronavir (show more...)Coronavirus disease-2019 (COVID-19), caused by the novel severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), has lead to a global pandemic with a rising toll in infections and deaths. Better understanding of its pathogenesis will greatly improve the outcomes and treatment of affected patients. Here we compared the inflammatory and cardiovascular disease-related protein cargo of circulating large and small extracellular vesicles (EVs) from 84 hospitalized patients infected with SARS-CoV-2 with different stages of disease severity. Our findings reveal significant enrichment of proinflammatory, procoagulation, immunoregulatory and tissue-remodelling protein signatures in EVs, which remarkably distinguished symptomatic COVID-19 patients from uninfected controls with matched comorbidities and delineated those with moderate disease from those who were critically ill. Specifically, EN-RAGE, followed by TF and IL-18R1, showed the strongest correlation with disease severity and length of hospitalization. Importantly, EVs from COVID-19 patients induced apoptosis of pulmonary microvascular endothelial cells in the order of disease severity. In conclusion, our findings support a role for EVs in the pathogenesis of COVID-19 disease and underpin the development of EV-based approaches to predicting disease severity, determining need for patient hospitalization and identifying new therapeutic targets. (hide) EV-METRIC 78% (98th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Blood plasma Sample origin COVID19 Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Protein markers EV: TSG101/ Alix/ CD63/ IL-18R1/ CD81/ IL-6RA/ Integrin beta1/ Flotillin1/ EN-RAGE/ CD9 non-EV: ApoE/ GM130 Proteomics no Show all info Study aim Function/Biomarker Sample Species Homo sapiens Sample Type Blood plasma Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 70 Pelleting: rotor type Surespin 630 (17 ml) Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 14 Wash: time (min) 70 Wash: Rotor Type Surespin 630 (17 ml) Wash: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method BCA Western Blot Detected EV-associated proteins Flotillin1/ Alix/ Integrin beta1/ TSG101/ CD9/ CD63/ CD81 Detected contaminants ApoE Not detected contaminants GM130 ELISA Detected EV-associated proteins EN-RAGE Flow cytometry Detected EV-associated proteins IL-6RA/ IL-18R1 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 50-300 EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 2.40E+10 EM EM-type Transmission-EM Image type Close-up, Wide-field Report size (nm) 30-200

	EV210153	2/11	Homo sapiens	22Rv1	(d)(U)C Filtration	Allelein, Susann	2021	78%
Study summary Full title Potential and challenges of specifically isolating extracellular vesicles from heterogeneous populations All authors Susann Allelein, Paula Medina-Perez, Ana Leonor Heitor Lopes, Sabrina Rau, Gerd Hause, Andreas Kölsch, Dirk Kuhlmeier Journal Sci Rep Abstract Extracellular vesicles (EVs) have attracted interest due to their ability to provide diagnostic info (show more...)Extracellular vesicles (EVs) have attracted interest due to their ability to provide diagnostic information from liquid biopsies. Cells constantly release vesicles divers in size, content and features depending on the biogenesis, origin and function. This heterogeneity adds a layer of complexity when attempting to isolate and characterize EVs resulting in various protocols. Their high abundance in all bodily fluids and their stable source of origin dependent biomarkers make EVs a powerful tool in biomarker discovery and diagnostics. However, applications are limited by the quality of samples definition. Here, we compared frequently used isolation techniques: ultracentrifugation, density gradient centrifugation, ultrafiltration and size exclusion chromatography. Then, we aimed for a tissue-specific isolation of prostate-derived EVs from cell culture supernatants with immunomagnetic beads. Quality and quantity of EVs were confirmed by nanoparticle tracking analysis, western blot and electron microscopy. Additionally, a spotted antibody microarray was developed to characterize EV sub-populations. Current analysis of 16 samples on one microarray for 6 different EV surface markers in triplicate could be easily extended allowing a faster and more economical method to characterize samples. (hide) EV-METRIC 78% (97th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Filtration Protein markers EV: TSG101/ Alix/ CD9 non-EV: Calnexin Proteomics no Show all info Study aim New methodological development/Biomarker/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells 22Rv1 EV-harvesting Medium Serum free medium Cell viability (%) 95 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type Surespin 630 (36 ml) Pelleting: speed (g) 110000 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method Fluorometric assay (e.g. Qubit, NanoOrange,...) Western Blot Detected EV-associated proteins CD9/ TSG101/ Alix Not detected contaminants Calnexin Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 140 EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 1.40E+11 EM EM-type Transmission electron microscopy Image type Close-up, Wide-field

	EV210143	1/6	Homo sapiens	SKBR3	(d)(U)C	Martinez-Pacheco, Sarai	2021	78%
Study summary Full title Evidence for the Need to Evaluate More Than One Source of Extracellular Vesicles, Rather Than Single or Pooled Samples Only, When Comparing Extracellular Vesicles Separation Methods All authors Sarai Martinez-Pacheco and Lorraine O’Driscoll Journal Cancers Abstract To study and exploit extracellular vesicles (EVs) for clinical benefit as biomarkers, therapeutics, (show more...)To study and exploit extracellular vesicles (EVs) for clinical benefit as biomarkers, therapeutics, or drug delivery vehicles in diseases such as cancer, typically we need to separate them from the biofluid into which they have been released by their cells of origin. For cultured cells, this fluid is conditioned medium (CM). Previous studies comparing EV separation approaches have typically focused on CM from one cell line or pooled samples of other biofluids. We hypothesize that this is inadequate and that extrapolating from a single source of EVs may not be informative. Thus, in our study of methods not previous compared (i.e., the original differential ultracentrifugation (dUC) method and a PEG followed by ultracentrifugation (PEG + UC) method), we analyzed CM from three different HER2-positive breast cancer cell lines (SKBR3, EFM192A, HCC1954) that grow in the same culture medium type. CM from each was collected and equally divided between both protocols. The resulting isolates were compared on seven characteristics/parameters including particle size, concentration, structure/morphology, protein content, purity, detection of five EV markers, and presence of HER2. Both dUC and PEG + UC generated reproducible data for any given breast cancer cell lines’ CM. However, the seven characteristics of the EV isolates were cell line- and method-dependent. This suggests the need to include more than one EV source, rather than a single or pooled sample, when selecting an EV separation method to be advanced for either research or clinical purposes (hide) EV-METRIC 78% (97th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Protein markers EV: CD63/ CD9/ Syntenin non-EV: Calnexin/ GRP94 Proteomics no Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells SKBR3 EV-harvesting Medium EV-depleted medium Preparation of EDS >=18h at >= 100,000g Cell viability (%) 94 Cell count 1.90E+08 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 70 Pelleting: rotor type Type 70 Ti Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 39 Wash: time (min) 70 Wash: Rotor Type Type 70 Ti Wash: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method microBCA Protein Yield (µg) Yes, per million cells 0.09 Western Blot Detected EV-associated proteins CD9/ CD63/ Syntenin Not detected contaminants Calnexin/ GRP94 Characterization: Lipid analysis Yes Characterization: Particle analysis NTA Report type Mean Reported size (nm) 125.7 EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 2296296296 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV210143	2/6	Homo sapiens	SKBR3	PEG precipitation (d)(U)C Filtration	Martinez-Pacheco, Sarai	2021	78%
Study summary Full title Evidence for the Need to Evaluate More Than One Source of Extracellular Vesicles, Rather Than Single or Pooled Samples Only, When Comparing Extracellular Vesicles Separation Methods All authors Sarai Martinez-Pacheco and Lorraine O’Driscoll Journal Cancers Abstract To study and exploit extracellular vesicles (EVs) for clinical benefit as biomarkers, therapeutics, (show more...)To study and exploit extracellular vesicles (EVs) for clinical benefit as biomarkers, therapeutics, or drug delivery vehicles in diseases such as cancer, typically we need to separate them from the biofluid into which they have been released by their cells of origin. For cultured cells, this fluid is conditioned medium (CM). Previous studies comparing EV separation approaches have typically focused on CM from one cell line or pooled samples of other biofluids. We hypothesize that this is inadequate and that extrapolating from a single source of EVs may not be informative. Thus, in our study of methods not previous compared (i.e., the original differential ultracentrifugation (dUC) method and a PEG followed by ultracentrifugation (PEG + UC) method), we analyzed CM from three different HER2-positive breast cancer cell lines (SKBR3, EFM192A, HCC1954) that grow in the same culture medium type. CM from each was collected and equally divided between both protocols. The resulting isolates were compared on seven characteristics/parameters including particle size, concentration, structure/morphology, protein content, purity, detection of five EV markers, and presence of HER2. Both dUC and PEG + UC generated reproducible data for any given breast cancer cell lines’ CM. However, the seven characteristics of the EV isolates were cell line- and method-dependent. This suggests the need to include more than one EV source, rather than a single or pooled sample, when selecting an EV separation method to be advanced for either research or clinical purposes (hide) EV-METRIC 78% (97th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture PEG precipitation (d)(U)C Filtration Protein markers EV: CD63/ CD9/ Syntenin non-EV: Calnexin/ GRP94 Proteomics no Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells SKBR3 EV-harvesting Medium EV-depleted medium Preparation of EDS >=18h at >= 100,000g Cell viability (%) 94 Cell count 1.90E+08 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 130 Pelleting: rotor type Type 70 Ti Pelleting: speed (g) 100000 Filtration steps 0.22µm or 0.2µm Other Name other separation method PEG precipitation Characterization: Protein analysis Protein Concentration Method microBCA Protein Yield (µg) Yes, per cell 0.14 Western Blot Detected EV-associated proteins CD9/ CD63/ Syntenin Not detected contaminants Calnexin/ GRP94 Characterization: Lipid analysis Yes Characterization: Particle analysis NTA Report type Mean Reported size (nm) 110.2 EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 6314814815 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV210143	3/6	Homo sapiens	HCC1954	(d)(U)C	Martinez-Pacheco, Sarai	2021	78%
Study summary Full title Evidence for the Need to Evaluate More Than One Source of Extracellular Vesicles, Rather Than Single or Pooled Samples Only, When Comparing Extracellular Vesicles Separation Methods All authors Sarai Martinez-Pacheco and Lorraine O’Driscoll Journal Cancers Abstract To study and exploit extracellular vesicles (EVs) for clinical benefit as biomarkers, therapeutics, (show more...)To study and exploit extracellular vesicles (EVs) for clinical benefit as biomarkers, therapeutics, or drug delivery vehicles in diseases such as cancer, typically we need to separate them from the biofluid into which they have been released by their cells of origin. For cultured cells, this fluid is conditioned medium (CM). Previous studies comparing EV separation approaches have typically focused on CM from one cell line or pooled samples of other biofluids. We hypothesize that this is inadequate and that extrapolating from a single source of EVs may not be informative. Thus, in our study of methods not previous compared (i.e., the original differential ultracentrifugation (dUC) method and a PEG followed by ultracentrifugation (PEG + UC) method), we analyzed CM from three different HER2-positive breast cancer cell lines (SKBR3, EFM192A, HCC1954) that grow in the same culture medium type. CM from each was collected and equally divided between both protocols. The resulting isolates were compared on seven characteristics/parameters including particle size, concentration, structure/morphology, protein content, purity, detection of five EV markers, and presence of HER2. Both dUC and PEG + UC generated reproducible data for any given breast cancer cell lines’ CM. However, the seven characteristics of the EV isolates were cell line- and method-dependent. This suggests the need to include more than one EV source, rather than a single or pooled sample, when selecting an EV separation method to be advanced for either research or clinical purposes (hide) EV-METRIC 78% (97th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Protein markers EV: CD63/ CD9/ Syntenin non-EV: Calnexin/ GRP94 Proteomics no Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells HCC1954 EV-harvesting Medium EV-depleted medium Preparation of EDS >=18h at >= 100,000g Cell viability (%) 93 Cell count 2.10E+08 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 70 Pelleting: rotor type Type 70 Ti Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 39 Wash: time (min) 70 Wash: Rotor Type Type 70 Ti Wash: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method microBCA Protein Yield (µg) Yes, per cell 0.03 Western Blot Detected EV-associated proteins CD9/ CD63/ Syntenin Not detected contaminants Calnexin/ GRP94 Characterization: Lipid analysis Yes Characterization: Particle analysis NTA Report type Mean Reported size (nm) 129.4 EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 7703703703 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV210143	4/6	Homo sapiens	HCC1954	PEG precipitation (d)(U)C Filtration	Martinez-Pacheco, Sarai	2021	78%
Study summary Full title Evidence for the Need to Evaluate More Than One Source of Extracellular Vesicles, Rather Than Single or Pooled Samples Only, When Comparing Extracellular Vesicles Separation Methods All authors Sarai Martinez-Pacheco and Lorraine O’Driscoll Journal Cancers Abstract To study and exploit extracellular vesicles (EVs) for clinical benefit as biomarkers, therapeutics, (show more...)To study and exploit extracellular vesicles (EVs) for clinical benefit as biomarkers, therapeutics, or drug delivery vehicles in diseases such as cancer, typically we need to separate them from the biofluid into which they have been released by their cells of origin. For cultured cells, this fluid is conditioned medium (CM). Previous studies comparing EV separation approaches have typically focused on CM from one cell line or pooled samples of other biofluids. We hypothesize that this is inadequate and that extrapolating from a single source of EVs may not be informative. Thus, in our study of methods not previous compared (i.e., the original differential ultracentrifugation (dUC) method and a PEG followed by ultracentrifugation (PEG + UC) method), we analyzed CM from three different HER2-positive breast cancer cell lines (SKBR3, EFM192A, HCC1954) that grow in the same culture medium type. CM from each was collected and equally divided between both protocols. The resulting isolates were compared on seven characteristics/parameters including particle size, concentration, structure/morphology, protein content, purity, detection of five EV markers, and presence of HER2. Both dUC and PEG + UC generated reproducible data for any given breast cancer cell lines’ CM. However, the seven characteristics of the EV isolates were cell line- and method-dependent. This suggests the need to include more than one EV source, rather than a single or pooled sample, when selecting an EV separation method to be advanced for either research or clinical purposes (hide) EV-METRIC 78% (97th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture PEG precipitation (d)(U)C Filtration Protein markers EV: CD63/ CD9/ Syntenin non-EV: Calnexin/ GRP94 Proteomics no Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells HCC1954 EV-harvesting Medium EV-depleted medium Preparation of EDS >=18h at >= 100,000g Cell viability (%) 93 Cell count 2.10E+08 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 130 Pelleting: rotor type Type 70 Ti Pelleting: speed (g) 100000 Filtration steps 0.22µm or 0.2µm Other Name other separation method PEG precipitation Characterization: Protein analysis Protein Concentration Method microBCA Protein Yield (µg) Yes, per cell 0.1 Western Blot Detected EV-associated proteins Syntenin/ CD9/ CD63 Not detected contaminants Calnexin/ GRP94 Characterization: Lipid analysis Yes Characterization: Particle analysis NTA Report type Mean Reported size (nm) 119.3 EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 8518481482 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV210143	5/6	Homo sapiens	EFM192A	(d)(U)C	Martinez-Pacheco, Sarai	2021	78%
Study summary Full title Evidence for the Need to Evaluate More Than One Source of Extracellular Vesicles, Rather Than Single or Pooled Samples Only, When Comparing Extracellular Vesicles Separation Methods All authors Sarai Martinez-Pacheco and Lorraine O’Driscoll Journal Cancers Abstract To study and exploit extracellular vesicles (EVs) for clinical benefit as biomarkers, therapeutics, (show more...)To study and exploit extracellular vesicles (EVs) for clinical benefit as biomarkers, therapeutics, or drug delivery vehicles in diseases such as cancer, typically we need to separate them from the biofluid into which they have been released by their cells of origin. For cultured cells, this fluid is conditioned medium (CM). Previous studies comparing EV separation approaches have typically focused on CM from one cell line or pooled samples of other biofluids. We hypothesize that this is inadequate and that extrapolating from a single source of EVs may not be informative. Thus, in our study of methods not previous compared (i.e., the original differential ultracentrifugation (dUC) method and a PEG followed by ultracentrifugation (PEG + UC) method), we analyzed CM from three different HER2-positive breast cancer cell lines (SKBR3, EFM192A, HCC1954) that grow in the same culture medium type. CM from each was collected and equally divided between both protocols. The resulting isolates were compared on seven characteristics/parameters including particle size, concentration, structure/morphology, protein content, purity, detection of five EV markers, and presence of HER2. Both dUC and PEG + UC generated reproducible data for any given breast cancer cell lines’ CM. However, the seven characteristics of the EV isolates were cell line- and method-dependent. This suggests the need to include more than one EV source, rather than a single or pooled sample, when selecting an EV separation method to be advanced for either research or clinical purposes (hide) EV-METRIC 78% (97th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Protein markers EV: CD63/ CD9/ Syntenin non-EV: Calnexin/ GRP94 Proteomics no Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells EFM192A EV-harvesting Medium EV-depleted medium Preparation of EDS >=18h at >= 100,000g Cell viability (%) 98 Cell count 3.60E+08 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 70 Pelleting: rotor type Type 70 Ti Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 39 Wash: time (min) 70 Wash: Rotor Type Type 70 Ti Wash: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method microBCA Protein Yield (µg) Yes, per cell 0.01 Western Blot Detected EV-associated proteins Syntenin/ CD63/ CD81 Not detected contaminants Calnexin/ GRP94 Characterization: Lipid analysis Yes Characterization: Particle analysis NTA Report type Mean Reported size (nm) 131.7 EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 727777778 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV210143	6/6	Homo sapiens	EFM192A	PEG precipitation (d)(U)C Filtration	Martinez-Pacheco, Sarai	2021	78%
Study summary Full title Evidence for the Need to Evaluate More Than One Source of Extracellular Vesicles, Rather Than Single or Pooled Samples Only, When Comparing Extracellular Vesicles Separation Methods All authors Sarai Martinez-Pacheco and Lorraine O’Driscoll Journal Cancers Abstract To study and exploit extracellular vesicles (EVs) for clinical benefit as biomarkers, therapeutics, (show more...)To study and exploit extracellular vesicles (EVs) for clinical benefit as biomarkers, therapeutics, or drug delivery vehicles in diseases such as cancer, typically we need to separate them from the biofluid into which they have been released by their cells of origin. For cultured cells, this fluid is conditioned medium (CM). Previous studies comparing EV separation approaches have typically focused on CM from one cell line or pooled samples of other biofluids. We hypothesize that this is inadequate and that extrapolating from a single source of EVs may not be informative. Thus, in our study of methods not previous compared (i.e., the original differential ultracentrifugation (dUC) method and a PEG followed by ultracentrifugation (PEG + UC) method), we analyzed CM from three different HER2-positive breast cancer cell lines (SKBR3, EFM192A, HCC1954) that grow in the same culture medium type. CM from each was collected and equally divided between both protocols. The resulting isolates were compared on seven characteristics/parameters including particle size, concentration, structure/morphology, protein content, purity, detection of five EV markers, and presence of HER2. Both dUC and PEG + UC generated reproducible data for any given breast cancer cell lines’ CM. However, the seven characteristics of the EV isolates were cell line- and method-dependent. This suggests the need to include more than one EV source, rather than a single or pooled sample, when selecting an EV separation method to be advanced for either research or clinical purposes (hide) EV-METRIC 78% (97th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture PEG precipitation (d)(U)C Filtration Protein markers EV: CD63/ CD9/ Syntenin non-EV: Calnexin/ GRP94 Proteomics no Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells EFM192A EV-harvesting Medium EV-depleted medium Preparation of EDS >=18h at >= 100,000g Cell viability (%) 98 Cell count 3.60E+08 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 130 Pelleting: rotor type Type 70 Ti Pelleting: speed (g) 100000 Filtration steps 0.22µm or 0.2µm Other Name other separation method PEG precipitation Characterization: Protein analysis Protein Concentration Method microBCA Protein Yield (µg) Yes, per cell 0.11 Western Blot Detected EV-associated proteins Syntenin/ CD63/ CD81 Not detected contaminants Calnexin/ GRP94 Characterization: Lipid analysis Yes Characterization: Particle analysis NTA Report type Mean Reported size (nm) 117.7 EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 5018518518 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV210126	1/9	Homo sapiens	Expi293F	(d)(U)C	Silva, Andreia;Lázaro-Ibáñez, Elisa	2021	78%
Study summary Full title Quantification of protein cargo loading into engineered extracellular vesicles at single-vesicle and single-molecule resolution All authors Andreia M. Silva, Elisa Lázaro-Ibáñez, Anders Gunnarsson, Aditya Dhande, George Daaboul, Ben Peacock, Xabier Osteikoetxea, Nikki Salmond, Kristina Pagh Friis, Olga Shatnyeva, Niek Dekker Journal J Extracell Vesicles Abstract Extracellular Vesicles (EVs) have been intensively explored for therapeutic delivery of proteins. Ho (show more...)Extracellular Vesicles (EVs) have been intensively explored for therapeutic delivery of proteins. However, methods to quantify cargo proteins loaded into engineered EVs are lacking. Here, we describe a workflow for EV analysis at the single-vesicle and single-molecule level to accurately quantify the efficiency of different EV-sorting proteins in promoting cargo loading into EVs. Expi293F cells were engineered to express EV-sorting proteins fused to green fluorescent protein (GFP). High levels of GFP loading into secreted EVs was confirmed by Western blotting for specific EV-sorting domains, but quantitative single-vesicle analysis by Nanoflow cytometry detected GFP in less than half of the particles analysed, reflecting EV heterogeneity. Anti-tetraspanin EV immunostaining in ExoView confirmed a heterogeneous GFP distribution in distinct subpopulations of CD63+, CD81+, or CD9+ EVs. Loading of GFP into individual vesicles was quantified by Single-Molecule Localization Microscopy. The combined results demonstrated TSPAN14, CD63 and CD63/CD81 fused to the PDGFRβ transmembrane domain as the most efficient EV-sorting proteins, accumulating on average 50–170 single GFP molecules per vesicle. In conclusion, we validated a set of complementary techniques suitable for high-resolution analysis of EV preparations that reliably capture their heterogeneity, and propose highly efficient EV-sorting proteins to be used in EV engineering applications. (hide) EV-METRIC 78% (97th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Protein markers EV: TSG101/ CD63/ CD81/ Alix/ Flotillin1/ CD9 non-EV: calnexin Proteomics no Show all info Study aim Biogenesis/cargo sorting/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Expi293F EV-harvesting Medium Serum free medium Cell viability (%) 80 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type Type 45 Ti Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 100 Wash: time (min) 120 Wash: Rotor Type Type 45 Ti Wash: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method Fluorometric assay (e.g. Qubit, NanoOrange,...) Western Blot Detected EV-associated proteins Flotillin1/ CD63/ GFP/ TSG101/ Alix/ CD81 Not detected EV-associated proteins CD9 Detected contaminants calnexin Flow cytometry Type of Flow cytometry NanoAnalyzer N30 (nanoFCM) Calibration bead size 0.068; 0.091; 0.113; 0.155 Detected EV-associated proteins CD9/ CD63/ CD81 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 100-200 EV concentration Yes Particle analysis: flow cytometry Flow cytometer type NanoAnalyzer N30 (nanoFCM) Hardware adjustment Calibration bead size 0.068; 0.091; 0.113; 0.155 Report type Mean Reported size (nm) 71 EV concentration Yes EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV210126	2/9	Homo sapiens	Expi293F	(d)(U)C	Silva, Andreia;Lázaro-Ibáñez, Elisa	2021	78%
Study summary Full title Quantification of protein cargo loading into engineered extracellular vesicles at single-vesicle and single-molecule resolution All authors Andreia M. Silva, Elisa Lázaro-Ibáñez, Anders Gunnarsson, Aditya Dhande, George Daaboul, Ben Peacock, Xabier Osteikoetxea, Nikki Salmond, Kristina Pagh Friis, Olga Shatnyeva, Niek Dekker Journal J Extracell Vesicles Abstract Extracellular Vesicles (EVs) have been intensively explored for therapeutic delivery of proteins. Ho (show more...)Extracellular Vesicles (EVs) have been intensively explored for therapeutic delivery of proteins. However, methods to quantify cargo proteins loaded into engineered EVs are lacking. Here, we describe a workflow for EV analysis at the single-vesicle and single-molecule level to accurately quantify the efficiency of different EV-sorting proteins in promoting cargo loading into EVs. Expi293F cells were engineered to express EV-sorting proteins fused to green fluorescent protein (GFP). High levels of GFP loading into secreted EVs was confirmed by Western blotting for specific EV-sorting domains, but quantitative single-vesicle analysis by Nanoflow cytometry detected GFP in less than half of the particles analysed, reflecting EV heterogeneity. Anti-tetraspanin EV immunostaining in ExoView confirmed a heterogeneous GFP distribution in distinct subpopulations of CD63+, CD81+, or CD9+ EVs. Loading of GFP into individual vesicles was quantified by Single-Molecule Localization Microscopy. The combined results demonstrated TSPAN14, CD63 and CD63/CD81 fused to the PDGFRβ transmembrane domain as the most efficient EV-sorting proteins, accumulating on average 50–170 single GFP molecules per vesicle. In conclusion, we validated a set of complementary techniques suitable for high-resolution analysis of EV preparations that reliably capture their heterogeneity, and propose highly efficient EV-sorting proteins to be used in EV engineering applications. (hide) EV-METRIC 78% (97th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin CD47-GFP Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Protein markers EV: TSG101/ CD63/ CD81/ Alix/ Flotillin1/ CD10 non-EV: calnexin Proteomics no Show all info Study aim Biogenesis/cargo sorting/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Expi293F EV-harvesting Medium Serum free medium Cell viability (%) 80 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type Type 45 Ti Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 100 Wash: time (min) 120 Wash: Rotor Type Type 45 Ti Wash: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method Fluorometric assay (e.g. Qubit, NanoOrange,...) Western Blot Detected EV-associated proteins Flotillin1/ CD63/ GFP/ TSG101/ Alix/ CD81 Not detected EV-associated proteins CD9 Detected contaminants calnexin Flow cytometry Type of Flow cytometry NanoAnalyzer N30 (nanoFCM) Calibration bead size 0.068; 0.091; 0.113; 0.155 Detected EV-associated proteins GFP Detected EV-associated proteins CD81/ CD9/ CD63/ GFP Detected EV-associated proteins GFP Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 100-200 EV concentration Yes Particle analysis: flow cytometry Flow cytometer type NanoAnalyzer N30 (nanoFCM) Hardware adjustment Calibration bead size 0.068; 0.091; 0.113; 0.155 Report type Mean Reported size (nm) 75 EV concentration Yes EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV210126	3/9	Homo sapiens	Expi293F	(d)(U)C	Silva, Andreia;Lázaro-Ibáñez, Elisa	2021	78%
Study summary Full title Quantification of protein cargo loading into engineered extracellular vesicles at single-vesicle and single-molecule resolution All authors Andreia M. Silva, Elisa Lázaro-Ibáñez, Anders Gunnarsson, Aditya Dhande, George Daaboul, Ben Peacock, Xabier Osteikoetxea, Nikki Salmond, Kristina Pagh Friis, Olga Shatnyeva, Niek Dekker Journal J Extracell Vesicles Abstract Extracellular Vesicles (EVs) have been intensively explored for therapeutic delivery of proteins. Ho (show more...)Extracellular Vesicles (EVs) have been intensively explored for therapeutic delivery of proteins. However, methods to quantify cargo proteins loaded into engineered EVs are lacking. Here, we describe a workflow for EV analysis at the single-vesicle and single-molecule level to accurately quantify the efficiency of different EV-sorting proteins in promoting cargo loading into EVs. Expi293F cells were engineered to express EV-sorting proteins fused to green fluorescent protein (GFP). High levels of GFP loading into secreted EVs was confirmed by Western blotting for specific EV-sorting domains, but quantitative single-vesicle analysis by Nanoflow cytometry detected GFP in less than half of the particles analysed, reflecting EV heterogeneity. Anti-tetraspanin EV immunostaining in ExoView confirmed a heterogeneous GFP distribution in distinct subpopulations of CD63+, CD81+, or CD9+ EVs. Loading of GFP into individual vesicles was quantified by Single-Molecule Localization Microscopy. The combined results demonstrated TSPAN14, CD63 and CD63/CD81 fused to the PDGFRβ transmembrane domain as the most efficient EV-sorting proteins, accumulating on average 50–170 single GFP molecules per vesicle. In conclusion, we validated a set of complementary techniques suitable for high-resolution analysis of EV preparations that reliably capture their heterogeneity, and propose highly efficient EV-sorting proteins to be used in EV engineering applications. (hide) EV-METRIC 78% (97th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin TSPAN14-GFP Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Protein markers EV: TSG101/ CD63/ CD81/ Alix/ Flotillin1/ CD11 non-EV: calnexin Proteomics no Show all info Study aim Biogenesis/cargo sorting/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Expi293F EV-harvesting Medium Serum free medium Cell viability (%) 80 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type Type 45 Ti Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 100 Wash: time (min) 120 Wash: Rotor Type Type 45 Ti Wash: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method Fluorometric assay (e.g. Qubit, NanoOrange,...) Western Blot Detected EV-associated proteins Flotillin1/ Alix/ CD63/ TSG101/ GFP/ CD81 Not detected EV-associated proteins CD9 Detected contaminants calnexin Flow cytometry Type of Flow cytometry NanoAnalyzer N30 (nanoFCM) Calibration bead size 0.068; 0.091; 0.113; 0.155 Detected EV-associated proteins GFP Detected EV-associated proteins CD81/ CD9/ CD63/ GFP Detected EV-associated proteins GFP Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 100-200 EV concentration Yes Particle analysis: flow cytometry Flow cytometer type NanoAnalyzer N30 (nanoFCM) Hardware adjustment Calibration bead size 0.068; 0.091; 0.113; 0.155 Report type Mean Reported size (nm) 73 EV concentration Yes EM EM-type Transmission-EM Image type Close-up, Wide-field

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