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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	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

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EV-TRACK ID	EV210118
species	Bos taurus
sample type	cheese manufacturing byproducts	cheese manufacturing byproducts	cheese manufacturing byproducts	skim milk
condition	Control condition	Control condition	Control condition	Control condition
separation protocol	DG tangential flow filtration Filtration	DG tangential flow filtration Filtration	DG tangential flow filtration Filtration	DG (d)(U)C
EV subtype	M.W. >100 kDa	M.W. 50 kDa to 100 kDa	M.W. >50 kDa	NA
Exp. nr.	1	2	3	4
EV-METRIC %	100	100	100	100