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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV190007	3/5	Homo sapiens	Urine	(d)(U)C Miltenyi Biotec Exosome Isolation Kit Pan UF	Mussack V	2019	75%
Study summary Full title Comparing small urinary extracellular vesicle purification methods with a view to RNA sequencing-Enabling robust and non-invasive biomarker research. All authors Mussack V, Wittmann G, Pfaffl MW. Journal Biomol Detect Quanti Abstract Small extracellular vesicles (EVs) are 50-200 nm sized mediators in intercellular communication th (show more...)Small extracellular vesicles (EVs) are 50-200 nm sized mediators in intercellular communication that reflect both physiological and pathophysiological changes of their parental cells. Thus, EVs hold great potential for biomarker detection. However, reliable purification methods for the downstream screening of the microRNA (miRNA) cargo carried within urinary EVs by small RNA sequencing have yet to be established. To address this knowledge gap, RNA extracted from human urinary EVs obtained by five different urinary EV purification methods (spin column chromatography, immunoaffinity, membrane affinity, precipitation and ultracentrifugation combined with density gradient) was analyzed by small RNA sequencing. Urinary EVs were further characterized by nanoparticle tracking analysis, Western blot analysis and transmission electron microscopy. Comprehensive EV characterization established significant method-dependent differences in size and concentration as well as variances in protein composition of isolated vesicles. Even though all purification methods captured enough total RNA to allow small RNA sequencing, method-dependent differences were also observed with respect to library sizes, mapping distributions, number of miRNA reads and diversity of transcripts. Whereas EVs obtained by immunoaffinity yielded the purest subset of small EVs, highly comparable with results attained by ultracentrifugation combined with density gradient, precipitation and membrane affinity, sample purification by spin column chromatography indicated a tendency to isolate different subtypes of small EVs, which might also carry a distinct subset of miRNAs. Based on our results, different EV purification methods seem to preferentially isolate different subtypes of EVs with varying efficiencies. As a consequence, sequencing experiments and resulting miRNA profiles were also affected. Hence, the selection of a specific EV isolation method has to satisfy the respective research question and should be well considered. In strict adherence with the MISEV (minimal information for studies of extracellular vesicles) guidelines, the importance of a combined evaluation of biophysical and proteomic EV characteristics alongside transcriptomic results was clearly demonstrated in this present study. (hide) EV-METRIC 75% (95th 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 Urine 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 Miltenyi Biotec Exosome Isolation Kit Pan UF Protein markers EV: TSG101/ CD63/ CD81/ Alix/ Syntenin/ EPCAM/ HSP70/ CD9 non-EV: Calnexin/ Tamm-Horsfall protein Proteomics no Show all info Study aim Identification of content (omics approaches)/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Urine Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Pelleting performed No Ultra filtration Cut-off size (kDa) 100 Membrane type Regenerated cellulose Commercial kit Other;Miltenyi Biotec Exosome Isolation Kit Pan Other Name other separation method Miltenyi Biotec Exosome Isolation Kit Pan Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? No Detected EV-associated proteins Alix/ CD9/ CD63/ TSG101/ Syntenin/ CD81 Not detected EV-associated proteins HSP70/ EPCAM Not detected contaminants Calnexin/ Tamm-Horsfall protein Characterization: RNA analysis RNA analysis Type RNAsequencing Database Yes Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 150 EV concentration Yes EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV190007	4/5	Homo sapiens	Urine	(d)(U)C Qiagen exoRNeasy Serum/Plasma Midi Kit UF	Mussack V	2019	75%
Study summary Full title Comparing small urinary extracellular vesicle purification methods with a view to RNA sequencing-Enabling robust and non-invasive biomarker research. All authors Mussack V, Wittmann G, Pfaffl MW. Journal Biomol Detect Quanti Abstract Small extracellular vesicles (EVs) are 50-200 nm sized mediators in intercellular communication th (show more...)Small extracellular vesicles (EVs) are 50-200 nm sized mediators in intercellular communication that reflect both physiological and pathophysiological changes of their parental cells. Thus, EVs hold great potential for biomarker detection. However, reliable purification methods for the downstream screening of the microRNA (miRNA) cargo carried within urinary EVs by small RNA sequencing have yet to be established. To address this knowledge gap, RNA extracted from human urinary EVs obtained by five different urinary EV purification methods (spin column chromatography, immunoaffinity, membrane affinity, precipitation and ultracentrifugation combined with density gradient) was analyzed by small RNA sequencing. Urinary EVs were further characterized by nanoparticle tracking analysis, Western blot analysis and transmission electron microscopy. Comprehensive EV characterization established significant method-dependent differences in size and concentration as well as variances in protein composition of isolated vesicles. Even though all purification methods captured enough total RNA to allow small RNA sequencing, method-dependent differences were also observed with respect to library sizes, mapping distributions, number of miRNA reads and diversity of transcripts. Whereas EVs obtained by immunoaffinity yielded the purest subset of small EVs, highly comparable with results attained by ultracentrifugation combined with density gradient, precipitation and membrane affinity, sample purification by spin column chromatography indicated a tendency to isolate different subtypes of small EVs, which might also carry a distinct subset of miRNAs. Based on our results, different EV purification methods seem to preferentially isolate different subtypes of EVs with varying efficiencies. As a consequence, sequencing experiments and resulting miRNA profiles were also affected. Hence, the selection of a specific EV isolation method has to satisfy the respective research question and should be well considered. In strict adherence with the MISEV (minimal information for studies of extracellular vesicles) guidelines, the importance of a combined evaluation of biophysical and proteomic EV characteristics alongside transcriptomic results was clearly demonstrated in this present study. (hide) EV-METRIC 75% (95th 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 Urine 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 Qiagen exoRNeasy Serum/Plasma Midi Kit UF Protein markers EV: TSG101/ CD63/ CD81/ Alix/ Syntenin/ EPCAM/ HSP70/ CD9 non-EV: Calnexin/ Tamm-Horsfall protein Proteomics no Show all info Study aim Identification of content (omics approaches)/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Urine Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Pelleting performed No Ultra filtration Cut-off size (kDa) 100 Membrane type Regenerated cellulose Commercial kit Other;Qiagen exoRNeasy Serum/Plasma Midi Kit Other Name other separation method Qiagen exoRNeasy Serum/Plasma Midi Kit Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? No Detected EV-associated proteins Alix/ Syntenin Not detected EV-associated proteins HSP70/ CD81/ EPCAM/ TSG101/ CD63/ CD9 Detected contaminants Tamm-Horsfall protein Not detected contaminants Calnexin Characterization: RNA analysis RNA analysis Type RNAsequencing Database Yes Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 120 EV concentration Yes EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV190007	5/5	Homo sapiens	Urine	(d)(U)C Exiqon miRCURY Exosome Isolation Kit UF	Mussack V	2019	75%
Study summary Full title Comparing small urinary extracellular vesicle purification methods with a view to RNA sequencing-Enabling robust and non-invasive biomarker research. All authors Mussack V, Wittmann G, Pfaffl MW. Journal Biomol Detect Quanti Abstract Small extracellular vesicles (EVs) are 50-200 nm sized mediators in intercellular communication th (show more...)Small extracellular vesicles (EVs) are 50-200 nm sized mediators in intercellular communication that reflect both physiological and pathophysiological changes of their parental cells. Thus, EVs hold great potential for biomarker detection. However, reliable purification methods for the downstream screening of the microRNA (miRNA) cargo carried within urinary EVs by small RNA sequencing have yet to be established. To address this knowledge gap, RNA extracted from human urinary EVs obtained by five different urinary EV purification methods (spin column chromatography, immunoaffinity, membrane affinity, precipitation and ultracentrifugation combined with density gradient) was analyzed by small RNA sequencing. Urinary EVs were further characterized by nanoparticle tracking analysis, Western blot analysis and transmission electron microscopy. Comprehensive EV characterization established significant method-dependent differences in size and concentration as well as variances in protein composition of isolated vesicles. Even though all purification methods captured enough total RNA to allow small RNA sequencing, method-dependent differences were also observed with respect to library sizes, mapping distributions, number of miRNA reads and diversity of transcripts. Whereas EVs obtained by immunoaffinity yielded the purest subset of small EVs, highly comparable with results attained by ultracentrifugation combined with density gradient, precipitation and membrane affinity, sample purification by spin column chromatography indicated a tendency to isolate different subtypes of small EVs, which might also carry a distinct subset of miRNAs. Based on our results, different EV purification methods seem to preferentially isolate different subtypes of EVs with varying efficiencies. As a consequence, sequencing experiments and resulting miRNA profiles were also affected. Hence, the selection of a specific EV isolation method has to satisfy the respective research question and should be well considered. In strict adherence with the MISEV (minimal information for studies of extracellular vesicles) guidelines, the importance of a combined evaluation of biophysical and proteomic EV characteristics alongside transcriptomic results was clearly demonstrated in this present study. (hide) EV-METRIC 75% (95th 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 Urine 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 Exiqon miRCURY Exosome Isolation Kit UF Protein markers EV: TSG101/ CD63/ CD81/ Alix/ Syntenin/ EPCAM/ HSP70/ CD9 non-EV: Calnexin/ Tamm-Horsfall protein Proteomics no Show all info Study aim Identification of content (omics approaches)/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Urine Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Pelleting performed No Ultra filtration Cut-off size (kDa) 100 Membrane type Regenerated cellulose Commercial kit Other;Exiqon miRCURY Exosome Isolation Kit Other Name other separation method Exiqon miRCURY Exosome Isolation Kit Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? No Detected EV-associated proteins CD9/ TSG101/ Alix/ CD81 Not detected EV-associated proteins HSP70/ EPCAM/ Syntenin/ CD63 Detected contaminants Tamm-Horsfall protein Not detected contaminants Calnexin Characterization: RNA analysis RNA analysis Type RNAsequencing Database Yes Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 145 EV concentration Yes EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV210502	7/8	Homo sapiens	Primary GBM Stem Cells JK2	(d)(U)C Filtration DG	Hallal S	2019	71%
Study summary Full title Extracellular Vesicles Released by Glioblastoma Cells Stimulate Normal Astrocytes to Acquire a Tumor-Supportive Phenotype Via p53 and MYC Signaling Pathways. All authors Hallal S, Mallawaaratchy DM, Wei H, Ebrahimkhani S, Stringer BW, Day BW, Boyd AW, Guillemin GJ, Buckland ME, Kaufman KL Journal Mol Neurobiol Abstract The role of astrocytes is becoming increasingly important to understanding how glioblastoma (GBM) tu (show more...)The role of astrocytes is becoming increasingly important to understanding how glioblastoma (GBM) tumor cells diffusely invade the brain. Yet, little is known of the contribution of extracellular vesicle (EV) signaling in GBM/astrocyte interactions. We modeled GBM-EV signaling to normal astrocytes in vitro to assess whether this mode of intercellular communication could support GBM progression. EVs were isolated and characterized from three patient-derived GBM stem cells (NES/CD133) and their differentiated (diff) progeny cells (NES/CD133). Uptake of GBM-EVs by normal primary astrocytes was confirmed by fluorescence microscopy, and changes in astrocyte podosome formation and gelatin degradation were measured. Quantitative mass spectrometry-based proteomics was performed on GBM-EV stimulated astrocytes. Interaction networks were generated from common, differentially abundant proteins using Ingenuity® (Qiagen Bioinformatics) and predicted upstream regulators were tested by qPCR assays. Podosome formation and Cy3-gelatin degradation were induced in astrocytes following 24-h exposure to GBM-stem and -diff EVs, with EVs released by GBM-stem cells eliciting a greater effect. More than 1700 proteins were quantified, and bioinformatics predicted activations of MYC, NFE2L2, FN1, and TGFβ1 and inhibition of TP53 in GBM-EV stimulated astrocytes that were then confirmed by qPCR. Further qPCR studies identified significantly decreased Δ133p53 and increased p53β in astrocytes exposed to GBM-EVs that might indicate the acquisition of a pro-inflammatory, tumor-promoting senescence-associated secretory phenotype (SASP). Inhibition of TP53 and activation of MYC signaling pathways in normal astrocytes exposed to GBM-EVs may be a mechanism by which GBM manipulates astrocytes to acquire a phenotype that promotes tumor progression. (hide) EV-METRIC 71% (96th 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 Filtration Density gradient Protein markers EV: None non-EV: None Proteomics yes Show all info Study aim Function/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Primary GBM Stem Cells JK2 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 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type Type 45 Ti Pelleting: speed (g) 100000 Wash: time (min) 180 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 4 Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 12.5 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 12 Pelleting: duration (min) 240 Pelleting: rotor type SW 41 Ti Pelleting: speed (g) 100000 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method Fluorometric assay (e.g. Qubit, NanoOrange,...) Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 140 EM EM-type Transmission-EM Image type Wide-field Report size (nm) 30-150

	EV210502	8/8	Homo sapiens	Primary GBM Stem Cells JK2	(d)(U)C Filtration DG	Hallal S	2019	71%
Study summary Full title Extracellular Vesicles Released by Glioblastoma Cells Stimulate Normal Astrocytes to Acquire a Tumor-Supportive Phenotype Via p53 and MYC Signaling Pathways. All authors Hallal S, Mallawaaratchy DM, Wei H, Ebrahimkhani S, Stringer BW, Day BW, Boyd AW, Guillemin GJ, Buckland ME, Kaufman KL Journal Mol Neurobiol Abstract The role of astrocytes is becoming increasingly important to understanding how glioblastoma (GBM) tu (show more...)The role of astrocytes is becoming increasingly important to understanding how glioblastoma (GBM) tumor cells diffusely invade the brain. Yet, little is known of the contribution of extracellular vesicle (EV) signaling in GBM/astrocyte interactions. We modeled GBM-EV signaling to normal astrocytes in vitro to assess whether this mode of intercellular communication could support GBM progression. EVs were isolated and characterized from three patient-derived GBM stem cells (NES/CD133) and their differentiated (diff) progeny cells (NES/CD133). Uptake of GBM-EVs by normal primary astrocytes was confirmed by fluorescence microscopy, and changes in astrocyte podosome formation and gelatin degradation were measured. Quantitative mass spectrometry-based proteomics was performed on GBM-EV stimulated astrocytes. Interaction networks were generated from common, differentially abundant proteins using Ingenuity® (Qiagen Bioinformatics) and predicted upstream regulators were tested by qPCR assays. Podosome formation and Cy3-gelatin degradation were induced in astrocytes following 24-h exposure to GBM-stem and -diff EVs, with EVs released by GBM-stem cells eliciting a greater effect. More than 1700 proteins were quantified, and bioinformatics predicted activations of MYC, NFE2L2, FN1, and TGFβ1 and inhibition of TP53 in GBM-EV stimulated astrocytes that were then confirmed by qPCR. Further qPCR studies identified significantly decreased Δ133p53 and increased p53β in astrocytes exposed to GBM-EVs that might indicate the acquisition of a pro-inflammatory, tumor-promoting senescence-associated secretory phenotype (SASP). Inhibition of TP53 and activation of MYC signaling pathways in normal astrocytes exposed to GBM-EVs may be a mechanism by which GBM manipulates astrocytes to acquire a phenotype that promotes tumor progression. (hide) EV-METRIC 71% (96th 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 Differentiated 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: None non-EV: None Proteomics yes Show all info Study aim Function/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Primary GBM Stem Cells JK2 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 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type Type 45 Ti Pelleting: speed (g) 100000 Wash: time (min) 180 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 4 Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 12.5 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 12 Pelleting: duration (min) 240 Pelleting: rotor type SW 41 Ti Pelleting: speed (g) 100000 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method Fluorometric assay (e.g. Qubit, NanoOrange,...) Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 127 EM EM-type Transmission-EM Image type Wide-field Report size (nm) 30-150

	EV180061	1/NA	Rattus norvegicus	NA	Filtration (d)(U)C ExoQuick	Lina Antounians	2019	67%
Study summary Full title The Regenerative Potential of Amniotic Fluid Stem Cell Extracellular Vesicles: Lessons Learned by Comparing Different Isolation Techniques All authors Lina Antounians, Areti Tzanetakis, Ornella Pellerito, Vincenzo D Catania, Adrienne Sulistyo, Louise Montalva, Mark J McVey, Augusto Zani Journal Sci Rep Abstract Extracellular vesicles (EVs) derived from amniotic fluid stem cells (AFSCs) mediate anti-apoptotic, (show more...)Extracellular vesicles (EVs) derived from amniotic fluid stem cells (AFSCs) mediate anti-apoptotic, pro-angiogenic, and immune-modulatory effects in multiple disease models, such as skeletal muscle atrophy and Alport syndrome. A source of potential variability in EV biological functions is how EV are isolated from parent cells. Currently, a comparative study of different EV isolation strategies using conditioned medium from AFSCs is lacking. Herein, we examined different isolation strategies for AFSC-EVs, using common techniques based on differential sedimentation (ultracentrifugation), solubility (ExoQuick, Total Exosome Isolation Reagent, Exo-PREP), or size-exclusion chromatography (qEV). All techniques isolated AFSC-EVs with typical EV morphology and protein markers. In contrast, AFSC-EV size, protein content, and yield varied depending on the method of isolation. When equal volumes of the different AFSC-EV preparations were used as treatment in a model of lung epithelial injury, we observed a significant variation in how AFSC-EVs were able to protect against cell death. AFSC-EV enhancement of cell survival appeared to be dose dependent, and largely uninfluenced by variation in EV-size distributions, relative EV-purity, or their total protein content. The variation in EV-mediated cell survival obtained with different isolation strategies emphasizes the importance of testing alternative isolation techniques in order to maximize EV regenerative capacity. (hide) EV-METRIC 67% (95th 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 NA 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 Filtration (Differential) (ultra)centrifugation Commercial method Protein markers EV: None non-EV: None Proteomics no Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Rattus norvegicus Sample Type NA EV-producing cells primary amniotic fluid stem cells 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 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 840 Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 100000 Commercial kit ExoQuick Characterization: Protein analysis Protein Concentration Method Bradford Western Blot Antibody details provided? No Detected EV-associated proteins HSP70/ TSG101/ Flotillin1/ CD63 Not detected contaminants histone marker Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 217.5 EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV180061	2/NA	Rattus norvegicus	NA	Filtration (d)(U)C Total Exosome Isolation	Lina Antounians	2019	67%
Study summary Full title The Regenerative Potential of Amniotic Fluid Stem Cell Extracellular Vesicles: Lessons Learned by Comparing Different Isolation Techniques All authors Lina Antounians, Areti Tzanetakis, Ornella Pellerito, Vincenzo D Catania, Adrienne Sulistyo, Louise Montalva, Mark J McVey, Augusto Zani Journal Sci Rep Abstract Extracellular vesicles (EVs) derived from amniotic fluid stem cells (AFSCs) mediate anti-apoptotic, (show more...)Extracellular vesicles (EVs) derived from amniotic fluid stem cells (AFSCs) mediate anti-apoptotic, pro-angiogenic, and immune-modulatory effects in multiple disease models, such as skeletal muscle atrophy and Alport syndrome. A source of potential variability in EV biological functions is how EV are isolated from parent cells. Currently, a comparative study of different EV isolation strategies using conditioned medium from AFSCs is lacking. Herein, we examined different isolation strategies for AFSC-EVs, using common techniques based on differential sedimentation (ultracentrifugation), solubility (ExoQuick, Total Exosome Isolation Reagent, Exo-PREP), or size-exclusion chromatography (qEV). All techniques isolated AFSC-EVs with typical EV morphology and protein markers. In contrast, AFSC-EV size, protein content, and yield varied depending on the method of isolation. When equal volumes of the different AFSC-EV preparations were used as treatment in a model of lung epithelial injury, we observed a significant variation in how AFSC-EVs were able to protect against cell death. AFSC-EV enhancement of cell survival appeared to be dose dependent, and largely uninfluenced by variation in EV-size distributions, relative EV-purity, or their total protein content. The variation in EV-mediated cell survival obtained with different isolation strategies emphasizes the importance of testing alternative isolation techniques in order to maximize EV regenerative capacity. (hide) EV-METRIC 67% (95th 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 NA 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 Filtration (Differential) (ultra)centrifugation Commercial method Protein markers EV: None non-EV: None Proteomics no Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Rattus norvegicus Sample Type NA EV-producing cells primary amniotic fluid stem cells 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 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 840 Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 100000 Commercial kit Total Exosome Isolation Characterization: Protein analysis Protein Concentration Method Bradford Western Blot Antibody details provided? No Detected EV-associated proteins HSP70/ TSG101/ Flotillin1/ CD63 Not detected contaminants histone marker Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 205.5 EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV180061	3/NA	Rattus norvegicus	NA	Filtration (d)(U)C Other;ExoPREP	Lina Antounians	2019	67%
Study summary Full title The Regenerative Potential of Amniotic Fluid Stem Cell Extracellular Vesicles: Lessons Learned by Comparing Different Isolation Techniques All authors Lina Antounians, Areti Tzanetakis, Ornella Pellerito, Vincenzo D Catania, Adrienne Sulistyo, Louise Montalva, Mark J McVey, Augusto Zani Journal Sci Rep Abstract Extracellular vesicles (EVs) derived from amniotic fluid stem cells (AFSCs) mediate anti-apoptotic, (show more...)Extracellular vesicles (EVs) derived from amniotic fluid stem cells (AFSCs) mediate anti-apoptotic, pro-angiogenic, and immune-modulatory effects in multiple disease models, such as skeletal muscle atrophy and Alport syndrome. A source of potential variability in EV biological functions is how EV are isolated from parent cells. Currently, a comparative study of different EV isolation strategies using conditioned medium from AFSCs is lacking. Herein, we examined different isolation strategies for AFSC-EVs, using common techniques based on differential sedimentation (ultracentrifugation), solubility (ExoQuick, Total Exosome Isolation Reagent, Exo-PREP), or size-exclusion chromatography (qEV). All techniques isolated AFSC-EVs with typical EV morphology and protein markers. In contrast, AFSC-EV size, protein content, and yield varied depending on the method of isolation. When equal volumes of the different AFSC-EV preparations were used as treatment in a model of lung epithelial injury, we observed a significant variation in how AFSC-EVs were able to protect against cell death. AFSC-EV enhancement of cell survival appeared to be dose dependent, and largely uninfluenced by variation in EV-size distributions, relative EV-purity, or their total protein content. The variation in EV-mediated cell survival obtained with different isolation strategies emphasizes the importance of testing alternative isolation techniques in order to maximize EV regenerative capacity. (hide) EV-METRIC 67% (95th 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 NA 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 Filtration (Differential) (ultra)centrifugation Commercial method Protein markers EV: None non-EV: None Proteomics no Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Rattus norvegicus Sample Type NA EV-producing cells primary amniotic fluid stem cells 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 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 840 Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 100000 Commercial kit Other;ExoPREP Characterization: Protein analysis Protein Concentration Method Bradford Western Blot Antibody details provided? No Detected EV-associated proteins HSP70/ TSG101/ Flotillin1/ CD63 Not detected contaminants histone marker Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 328.8 EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV180061	4/NA	Rattus norvegicus	NA	Filtration (d)(U)C qEV	Lina Antounians	2019	67%
Study summary Full title The Regenerative Potential of Amniotic Fluid Stem Cell Extracellular Vesicles: Lessons Learned by Comparing Different Isolation Techniques All authors Lina Antounians, Areti Tzanetakis, Ornella Pellerito, Vincenzo D Catania, Adrienne Sulistyo, Louise Montalva, Mark J McVey, Augusto Zani Journal Sci Rep Abstract Extracellular vesicles (EVs) derived from amniotic fluid stem cells (AFSCs) mediate anti-apoptotic, (show more...)Extracellular vesicles (EVs) derived from amniotic fluid stem cells (AFSCs) mediate anti-apoptotic, pro-angiogenic, and immune-modulatory effects in multiple disease models, such as skeletal muscle atrophy and Alport syndrome. A source of potential variability in EV biological functions is how EV are isolated from parent cells. Currently, a comparative study of different EV isolation strategies using conditioned medium from AFSCs is lacking. Herein, we examined different isolation strategies for AFSC-EVs, using common techniques based on differential sedimentation (ultracentrifugation), solubility (ExoQuick, Total Exosome Isolation Reagent, Exo-PREP), or size-exclusion chromatography (qEV). All techniques isolated AFSC-EVs with typical EV morphology and protein markers. In contrast, AFSC-EV size, protein content, and yield varied depending on the method of isolation. When equal volumes of the different AFSC-EV preparations were used as treatment in a model of lung epithelial injury, we observed a significant variation in how AFSC-EVs were able to protect against cell death. AFSC-EV enhancement of cell survival appeared to be dose dependent, and largely uninfluenced by variation in EV-size distributions, relative EV-purity, or their total protein content. The variation in EV-mediated cell survival obtained with different isolation strategies emphasizes the importance of testing alternative isolation techniques in order to maximize EV regenerative capacity. (hide) EV-METRIC 67% (95th 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 NA 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 Filtration (Differential) (ultra)centrifugation Commercial method Protein markers EV: None non-EV: None Proteomics no Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Rattus norvegicus Sample Type NA EV-producing cells primary amniotic fluid stem cells 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 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 840 Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 100000 Commercial kit qEV Characterization: Protein analysis Protein Concentration Method Bradford Western Blot Antibody details provided? No Detected EV-associated proteins HSP70 Not detected EV-associated proteins CD63/ Flotillin1/ TSG101 Not detected contaminants histone marker Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 53 EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV180061	5/NA	Rattus norvegicus	NA	Filtration (d)(U)C	Lina Antounians	2019	67%
Study summary Full title The Regenerative Potential of Amniotic Fluid Stem Cell Extracellular Vesicles: Lessons Learned by Comparing Different Isolation Techniques All authors Lina Antounians, Areti Tzanetakis, Ornella Pellerito, Vincenzo D Catania, Adrienne Sulistyo, Louise Montalva, Mark J McVey, Augusto Zani Journal Sci Rep Abstract Extracellular vesicles (EVs) derived from amniotic fluid stem cells (AFSCs) mediate anti-apoptotic, (show more...)Extracellular vesicles (EVs) derived from amniotic fluid stem cells (AFSCs) mediate anti-apoptotic, pro-angiogenic, and immune-modulatory effects in multiple disease models, such as skeletal muscle atrophy and Alport syndrome. A source of potential variability in EV biological functions is how EV are isolated from parent cells. Currently, a comparative study of different EV isolation strategies using conditioned medium from AFSCs is lacking. Herein, we examined different isolation strategies for AFSC-EVs, using common techniques based on differential sedimentation (ultracentrifugation), solubility (ExoQuick, Total Exosome Isolation Reagent, Exo-PREP), or size-exclusion chromatography (qEV). All techniques isolated AFSC-EVs with typical EV morphology and protein markers. In contrast, AFSC-EV size, protein content, and yield varied depending on the method of isolation. When equal volumes of the different AFSC-EV preparations were used as treatment in a model of lung epithelial injury, we observed a significant variation in how AFSC-EVs were able to protect against cell death. AFSC-EV enhancement of cell survival appeared to be dose dependent, and largely uninfluenced by variation in EV-size distributions, relative EV-purity, or their total protein content. The variation in EV-mediated cell survival obtained with different isolation strategies emphasizes the importance of testing alternative isolation techniques in order to maximize EV regenerative capacity. (hide) EV-METRIC 67% (95th 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 NA 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 Filtration (Differential) (ultra)centrifugation Protein markers EV: None non-EV: None Proteomics no Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Rattus norvegicus Sample Type NA EV-producing cells primary amniotic fluid stem cells 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 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 840 Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? No Detected EV-associated proteins TSG101/ HSP70/ Flotillin1/ CD63 Not detected contaminants histone marker Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 181.8 EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV200028	1/3	Rattus norvegicus	Serum	(d)(U)C	F Fricke	2019	67%
Study summary Full title Proinflammatory Extracellular Vesicle-Mediated Signaling Contributes to the Induction of Neuroinflammation in Animal Models of Endotoxemia and Peripheral Surgical Stress All authors F Fricke, J Gebert, J Kopitz, K Plaschke Journal Cell Mol Neurobiol Abstract Peripheral inflammation induced by endotoxemia or surgical stress induces neuroinflammation thereby (show more...)Peripheral inflammation induced by endotoxemia or surgical stress induces neuroinflammation thereby causing neurological symptoms ranging from sickness behavior to delirium. Thus, proinflammatory signaling must be operative between the periphery and the central nervous system (CNS). In the present study, we tested whether nanometer-sized extracellular vesicles (EVs) that were produced during the peripheral inflammatory process have the capacity to induce neuroinflammation. Conditions of endotoxemia or surgical intervention were simulated in rats by lipopolysaccharide (LPS) injection or partial hepatectomy (HpX). EVs were concentrated from these animals and tested for their proinflammatory action (I) in a microglial cell line and (II) by intracerebroventricular and (III) by intravenous injections into healthy rats. EVs from both conditions induced the secretion of cytokines from the glial cell line. Intracerebroventricular injection of the EVs caused the release of inflammatory cytokines to the cerebrospinal fluid indicating their pro-neuroinflammatory capacity. Finally, proinflammatory EVs were shown to pass the blood-brain barrier and induce neuroinflammation after their intravenous injection. Based on these data, we suggest that EV-associated proinflammatory signaling contributes to the induction of neuroinflammation in endotoxemia and peripheral surgical stress. Preliminary results suggest that peripheral cholinergic signals might be involved in the control of proinflammatory EV-mediated signaling from the periphery to the brain. (hide) EV-METRIC 67% (96th 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 Serum 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: Alix/ CD63/ CD9 non-EV: ApoA1 Proteomics no Show all info Study aim Function/Mechanism of uptake/transfer Sample Species Rattus norvegicus Sample Type Serum 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) 120 Pelleting: rotor type TLA-100.2 Pelleting: speed (g) 120000 Characterization: Protein analysis Protein Concentration Method Bradford Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD9/ CD63/ Alix Not detected contaminants ApoA1 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 107 EM EM-type Transmission-EM Image type Wide-field

	EV200028	2/3	Rattus norvegicus	Serum	(d)(U)C	F Fricke	2019	67%
Study summary Full title Proinflammatory Extracellular Vesicle-Mediated Signaling Contributes to the Induction of Neuroinflammation in Animal Models of Endotoxemia and Peripheral Surgical Stress All authors F Fricke, J Gebert, J Kopitz, K Plaschke Journal Cell Mol Neurobiol Abstract Peripheral inflammation induced by endotoxemia or surgical stress induces neuroinflammation thereby (show more...)Peripheral inflammation induced by endotoxemia or surgical stress induces neuroinflammation thereby causing neurological symptoms ranging from sickness behavior to delirium. Thus, proinflammatory signaling must be operative between the periphery and the central nervous system (CNS). In the present study, we tested whether nanometer-sized extracellular vesicles (EVs) that were produced during the peripheral inflammatory process have the capacity to induce neuroinflammation. Conditions of endotoxemia or surgical intervention were simulated in rats by lipopolysaccharide (LPS) injection or partial hepatectomy (HpX). EVs were concentrated from these animals and tested for their proinflammatory action (I) in a microglial cell line and (II) by intracerebroventricular and (III) by intravenous injections into healthy rats. EVs from both conditions induced the secretion of cytokines from the glial cell line. Intracerebroventricular injection of the EVs caused the release of inflammatory cytokines to the cerebrospinal fluid indicating their pro-neuroinflammatory capacity. Finally, proinflammatory EVs were shown to pass the blood-brain barrier and induce neuroinflammation after their intravenous injection. Based on these data, we suggest that EV-associated proinflammatory signaling contributes to the induction of neuroinflammation in endotoxemia and peripheral surgical stress. Preliminary results suggest that peripheral cholinergic signals might be involved in the control of proinflammatory EV-mediated signaling from the periphery to the brain. (hide) EV-METRIC 67% (96th 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 Serum Sample origin LPS 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: Alix/ CD63/ CD9 non-EV: ApoA1 Proteomics no Show all info Study aim Function/Mechanism of uptake/transfer Sample Species Rattus norvegicus Sample Type Serum 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) 120 Pelleting: rotor type TLA-100.2 Pelleting: speed (g) 120000 Characterization: Protein analysis Protein Concentration Method Bradford Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD9/ CD63/ Alix Not detected contaminants ApoA1 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 106 EM EM-type Transmission-EM Image type Wide-field

	EV200028	3/3	Rattus norvegicus	Serum	(d)(U)C	F Fricke	2019	67%
Study summary Full title Proinflammatory Extracellular Vesicle-Mediated Signaling Contributes to the Induction of Neuroinflammation in Animal Models of Endotoxemia and Peripheral Surgical Stress All authors F Fricke, J Gebert, J Kopitz, K Plaschke Journal Cell Mol Neurobiol Abstract Peripheral inflammation induced by endotoxemia or surgical stress induces neuroinflammation thereby (show more...)Peripheral inflammation induced by endotoxemia or surgical stress induces neuroinflammation thereby causing neurological symptoms ranging from sickness behavior to delirium. Thus, proinflammatory signaling must be operative between the periphery and the central nervous system (CNS). In the present study, we tested whether nanometer-sized extracellular vesicles (EVs) that were produced during the peripheral inflammatory process have the capacity to induce neuroinflammation. Conditions of endotoxemia or surgical intervention were simulated in rats by lipopolysaccharide (LPS) injection or partial hepatectomy (HpX). EVs were concentrated from these animals and tested for their proinflammatory action (I) in a microglial cell line and (II) by intracerebroventricular and (III) by intravenous injections into healthy rats. EVs from both conditions induced the secretion of cytokines from the glial cell line. Intracerebroventricular injection of the EVs caused the release of inflammatory cytokines to the cerebrospinal fluid indicating their pro-neuroinflammatory capacity. Finally, proinflammatory EVs were shown to pass the blood-brain barrier and induce neuroinflammation after their intravenous injection. Based on these data, we suggest that EV-associated proinflammatory signaling contributes to the induction of neuroinflammation in endotoxemia and peripheral surgical stress. Preliminary results suggest that peripheral cholinergic signals might be involved in the control of proinflammatory EV-mediated signaling from the periphery to the brain. (hide) EV-METRIC 67% (96th 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 Serum Sample origin HpX 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: Alix/ CD63/ CD9 non-EV: ApoA1 Proteomics no Show all info Study aim Function/Mechanism of uptake/transfer Sample Species Rattus norvegicus Sample Type Serum 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) 120 Pelleting: rotor type TLA-100.2 Pelleting: speed (g) 120000 Characterization: Protein analysis Protein Concentration Method Bradford Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins Alix/ CD9/ CD63 Not detected contaminants ApoA1 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 109 EM EM-type Transmission-EM Image type Wide-field

	EV190098	1/3	Mus musculus	MC-38	(d)(U)C	Izabela Papiewska-Pająk	2019	67%
Study summary Full title Glypican-1 Level Is Elevated in Extracellular Vesicles Released from MC38 Colon Adenocarcinoma Cells Overexpressing Snail All authors Izabela Papiewska-Pająk, Damian Krzyżanowski, Maria Katela, Romain Rivet, Sylwia Michlewska, Patrycja Przygodzka, M Anna Kowalska, Stéphane Brézillon Journal Cells Abstract The transcription factor Snail triggers epithelial-to-mesenchymal transition (EMT), endowing cancer (show more...)The transcription factor Snail triggers epithelial-to-mesenchymal transition (EMT), endowing cancer cells with invasive properties during tumor progression. Extracellular vesicles (EVs) released from cancer cells at various stages of cancer progression are known to influence the tumor pre-metastatic niche and metastatic potential. The aim of this study was to analyze the effect of Snail on murine colon adenocarcinoma cells (MC38 line) and on the characteristics of their EVs. Stable clones of Snail-overexpressing MC38 cells were investigated in vitro versus Mock cells. Increased expression of matrix metalloproteinase MMP-14 and augmented activity of MMP-9 and -14 were observed in Snail-MC38 cells. There was no change in the transcriptomic profile of proteoglycans in Snail-MC38 cells; however, the protein level of Glypican-1 (GPC1) was enhanced in EVs released from those cells. Our finding that GPC1 protein level was enhanced in EVs released from MC38 cells that overexpressed Snail and were in an early EMT stage might explain the specificity of the GPC1 biomarker in colon cancer diagnosis. Further, our data suggest that Snail, by changing the level of GPC1 on EVs released by colon cancer cells, may affect the generation of a distant premetastatic niche and metastatic organotropism in colon adenocarcinoma. (hide) EV-METRIC 67% (94th 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 MC38-pcDNA 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: ANXA5/ HSP70/ Alix non-EV: cytochrome c Proteomics no Show all info Study aim Function/Identification of content (omics approaches) Sample Species Mus musculus Sample Type Cell culture supernatant EV-producing cells MC-38 EV-harvesting Medium Serum free medium Cell viability (%) 97 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) 90 Pelleting: rotor type Type 45 Ti Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 22.5 Wash: time (min) 90 Wash: Rotor Type Type 45 Ti Wash: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins ANXA5/ HSP70/ Alix Not detected contaminants cytochrome c Characterization: Lipid analysis No Characterization: Particle analysis DLS Report type Size range/distribution Reported size (nm) 100-500 EM EM-type Transmission-EM Image type Wide-field

	EV190098	2/3	Mus musculus	MC-38	(d)(U)C	Izabela Papiewska-Pająk	2019	67%
Study summary Full title Glypican-1 Level Is Elevated in Extracellular Vesicles Released from MC38 Colon Adenocarcinoma Cells Overexpressing Snail All authors Izabela Papiewska-Pająk, Damian Krzyżanowski, Maria Katela, Romain Rivet, Sylwia Michlewska, Patrycja Przygodzka, M Anna Kowalska, Stéphane Brézillon Journal Cells Abstract The transcription factor Snail triggers epithelial-to-mesenchymal transition (EMT), endowing cancer (show more...)The transcription factor Snail triggers epithelial-to-mesenchymal transition (EMT), endowing cancer cells with invasive properties during tumor progression. Extracellular vesicles (EVs) released from cancer cells at various stages of cancer progression are known to influence the tumor pre-metastatic niche and metastatic potential. The aim of this study was to analyze the effect of Snail on murine colon adenocarcinoma cells (MC38 line) and on the characteristics of their EVs. Stable clones of Snail-overexpressing MC38 cells were investigated in vitro versus Mock cells. Increased expression of matrix metalloproteinase MMP-14 and augmented activity of MMP-9 and -14 were observed in Snail-MC38 cells. There was no change in the transcriptomic profile of proteoglycans in Snail-MC38 cells; however, the protein level of Glypican-1 (GPC1) was enhanced in EVs released from those cells. Our finding that GPC1 protein level was enhanced in EVs released from MC38 cells that overexpressed Snail and were in an early EMT stage might explain the specificity of the GPC1 biomarker in colon cancer diagnosis. Further, our data suggest that Snail, by changing the level of GPC1 on EVs released by colon cancer cells, may affect the generation of a distant premetastatic niche and metastatic organotropism in colon adenocarcinoma. (hide) EV-METRIC 67% (94th 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 MC38-Snail 2 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: ANXA5/ HSP70/ Alix non-EV: cytochrome c Proteomics no Show all info Study aim Function/Identification of content (omics approaches) Sample Species Mus musculus Sample Type Cell culture supernatant EV-producing cells MC-38 EV-harvesting Medium Serum free medium Cell viability (%) 97 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) 90 Pelleting: rotor type Type 45 Ti Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 22.5 Wash: time (min) 90 Wash: Rotor Type Type 45 Ti Wash: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins ANXA5/ HSP70/ Alix Not detected contaminants cytochrome c Characterization: Lipid analysis No EM EM-type Transmission-EM Image type Wide-field

	EV190098	3/3	Mus musculus	MC-38	(d)(U)C	Izabela Papiewska-Pająk	2019	67%
Study summary Full title Glypican-1 Level Is Elevated in Extracellular Vesicles Released from MC38 Colon Adenocarcinoma Cells Overexpressing Snail All authors Izabela Papiewska-Pająk, Damian Krzyżanowski, Maria Katela, Romain Rivet, Sylwia Michlewska, Patrycja Przygodzka, M Anna Kowalska, Stéphane Brézillon Journal Cells Abstract The transcription factor Snail triggers epithelial-to-mesenchymal transition (EMT), endowing cancer (show more...)The transcription factor Snail triggers epithelial-to-mesenchymal transition (EMT), endowing cancer cells with invasive properties during tumor progression. Extracellular vesicles (EVs) released from cancer cells at various stages of cancer progression are known to influence the tumor pre-metastatic niche and metastatic potential. The aim of this study was to analyze the effect of Snail on murine colon adenocarcinoma cells (MC38 line) and on the characteristics of their EVs. Stable clones of Snail-overexpressing MC38 cells were investigated in vitro versus Mock cells. Increased expression of matrix metalloproteinase MMP-14 and augmented activity of MMP-9 and -14 were observed in Snail-MC38 cells. There was no change in the transcriptomic profile of proteoglycans in Snail-MC38 cells; however, the protein level of Glypican-1 (GPC1) was enhanced in EVs released from those cells. Our finding that GPC1 protein level was enhanced in EVs released from MC38 cells that overexpressed Snail and were in an early EMT stage might explain the specificity of the GPC1 biomarker in colon cancer diagnosis. Further, our data suggest that Snail, by changing the level of GPC1 on EVs released by colon cancer cells, may affect the generation of a distant premetastatic niche and metastatic organotropism in colon adenocarcinoma. (hide) EV-METRIC 67% (94th 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 MC38-Snail 6 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: ANXA5/ HSP70/ Alix non-EV: cytochrome c Proteomics no Show all info Study aim Function/Identification of content (omics approaches) Sample Species Mus musculus Sample Type Cell culture supernatant EV-producing cells MC-38 EV-harvesting Medium Serum free medium Cell viability (%) 97 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) 90 Pelleting: rotor type Type 45 Ti Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 22.5 Wash: time (min) 90 Wash: Rotor Type Type 45 Ti Wash: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins ANXA5/ HSP70/ Alix Not detected contaminants cytochrome c Characterization: Lipid analysis No EM EM-type Transmission-EM Image type Wide-field

	EV190090	1/1	Homo sapiens	HCT116	(d)(U)C Filtration	Torres Crigna A	2019	67%
Study summary Full title Inter-Laboratory Comparison of Extracellular Vesicle Isolation Based on Ultracentrifugation All authors Torres Crigna A., Fricke F., Nitschke K., Worst T., Erb U., Karremann M., Buschmann D., Elvers-Hornung S., Tucher C., Schiller M., Hausser I., Gebert J., Bieback K. Journal Transfus Med Hemother Abstract Background/Aims: Extracellular vesicles (EVs), including microvesicles and exosomes, deliver bioacti (show more...)Background/Aims: Extracellular vesicles (EVs), including microvesicles and exosomes, deliver bioactive cargo mediating intercellular communication in physiological and pathological conditions. EVs are increasingly investigated as therapeutic agents and targets, but also as disease biomarkers. However, a definite consensus regarding EV isolation methods is lacking, which makes it intricate to standardize research practices and eventually reach a desirable level of data comparability. In our study, we performed an inter-laboratory comparison of EV isolation based on a differential ultracentrifugation protocol carried out in 4 laboratories in 2 independent rounds of isolation. Methods: Conditioned medium of colorectal cancer cells was prepared and pooled by 1 person and distributed to each of the participating laboratories for isolation according to a pre-defined protocol. After EV isolation in each laboratory, quantification and characterization of isolated EVs was collectively done by 1 person having the highest expertise in the respective test method: Western blot, flow cytometry (fluorescence-activated cell sorting [FACS], nanoparticle tracking analysis (NTA), and transmission electron microscopy (TEM). Results: EVs were visualized with TEM, presenting similar cup-shaped and spherical morphology and sizes ranging from 30 to 150 nm. NTA results showed similar size ranges of particles in both isolation rounds. EV preparations showed high purity by the expression of EV marker proteins CD9, CD63, CD81, Alix, and TSG101, and the lack of calnexin. FACS analysis of EVs revealed intense staining for CD63 and CD81 but lower levels for CD9 and TSG101. Preparations from 1 laboratory presented significantly lower particle numbers (p < 0.0001), most probably related to increased processing time. However, even when standardizing processing time, particle yields still differed significantly between groups, indicating inter-laboratory differences in the efficiency of EV isolation. Importantly, no relation was observed between centrifugation speed/k-factor and EV yield. Conclusions: Our findings demonstrate that quantitative differences in EV yield might be due to equipment- and operator-dependent technical variability in ultracentrifugation-based EV isolation. Furthermore, our study emphasizes the need to standardize technical parameters such as the exact run speed and k-factor in order to transfer protocols between different laboratories. This hints at substantial inter-laboratory biases that should be assessed in multi-centric studies. (hide) EV-METRIC 67% (94th 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/ CD81/ CD63/ CD9/ Alix non-EV: calnexin 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 HCT116 EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g 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 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 Surespin 630 (17 ml) Pelleting: speed (g) 23000 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD9/ CD63/ TSG101/ Alix/ CD81 Not detected contaminants calnexin Flow cytometry specific beads Antibody details provided? No Antibody dilution provided? No Detected EV-associated proteins CD9/ CD63/ TSG101/ CD81 Not detected EV-associated proteins Alix Not detected contaminants calnexin Characterization: Lipid analysis No EM EM-type Transmission-EM Image type Close-up Report size (nm) 20-180

	EV190058	1/1	Homo sapiens	primary spindle-shaped amniotic fluid stem cells	(d)(U)C qEV UF	Takov K	2019	67%
Study summary Full title Small extracellular vesicles secreted from human amniotic fluid mesenchymal stromal cells possess cardioprotective and promigratory potential. All authors Takov K, He Z, Johnston HE, Timms JF, Guillot PV, Yellon DM, Davidson SM Journal Basic Res Cardiol Abstract Mesenchymal stromal cells (MSCs) exhibit antiapoptotic and proangiogenic functions in models of myoc (show more...)Mesenchymal stromal cells (MSCs) exhibit antiapoptotic and proangiogenic functions in models of myocardial infarction which may be mediated by secreted small extracellular vesicles (sEVs). However, MSCs have frequently been harvested from aged or diseased patients, while the isolated sEVs often contain high levels of impurities. Here, we studied the cardioprotective and proangiogenic activities of size-exclusion chromatography-purified sEVs secreted from human foetal amniotic fluid stem cells (SS-hAFSCs), possessing superior functional potential to that of adult MSCs. We demonstrated for the first time that highly pure (up to 1.7 × 1010 particles/µg protein) and thoroughly characterised SS-hAFSC sEVs protect rat hearts from ischaemia-reperfusion injury in vivo when administered intravenously prior to reperfusion (38 ± 9% infarct size reduction, p < 0.05). SS-hAFSC sEVs did not protect isolated primary cardiomyocytes in models of simulated ischaemia-reperfusion injury in vitro, indicative of indirect cardioprotective effects. SS-hAFSC sEVs were not proangiogenic in vitro, although they markedly stimulated endothelial cell migration. Additionally, sEVs were entirely responsible for the promigratory effects of the medium conditioned by SS-hAFSC. Mechanistically, sEV-induced chemotaxis involved phosphatidylinositol 3-kinase (PI3K) signalling, as its pharmacological inhibition in treated endothelial cells reduced migration by 54 ± 7% (p < 0.001). Together, these data indicate that SS-hAFSC sEVs have multifactorial beneficial effects in a myocardial infarction setting. (hide) EV-METRIC 67% (94th 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 small extracellular vesicles (sEVs) 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 qEV UF Protein markers EV: CD81/ CD63/ CD9 non-EV: ACTN4 Proteomics yes Show all info Study aim Function/Identification of content (omics approaches)/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells primary spindle-shaped amniotic fluid stem cells EV-harvesting Medium Serum free medium Cell viability (%) 86 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 10,000 g and 50,000 g Pelleting performed No Ultra filtration Cut-off size (kDa) 30 Membrane type Hydrosart (stabilised cellulose);Other Commercial kit qEV Other Name other separation method qEV EV-subtype Distinction between multiple subtypes Used subtypes Characterization: Protein analysis Protein Concentration Method microBCA;Bradford Proteomics database No Other 1 Immunoassay (DELFIA) Detected EV-associated proteins CD9/ CD63/ CD81 Other 2 Dot blot Detected EV-associated proteins CD81/ CD63 Not detected EV-associated proteins CD9 Not detected contaminants ACTN4 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mode Reported size (nm) 100.5 EV concentration Yes EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV190055	2/4	Homo sapiens	ARPE-19	DG (d)(U)C	Ferreira JV	2019	67%
Study summary Full title Exosomes and STUB1/CHIP cooperate to maintain intracellular proteostasis. All authors Ferreira JV, Rosa Soares A, Ramalho JS, Ribeiro-Rodrigues T, Máximo C, Zuzarte M, Girão H, Pereira P. Journal PLoS One Abstract Deregulation of proteostasis is a main feature of many age-related diseases, often leading to the ac (show more...)Deregulation of proteostasis is a main feature of many age-related diseases, often leading to the accumulation of toxic oligomers and insoluble protein aggregates that accumulate intracellularly or in the extracellular space. To understand the mechanisms whereby toxic or otherwise unwanted proteins are secreted to the extracellular space, we inactivated the quality-control and proteostasis regulator ubiquitin ligase STUB1/CHIP. Data indicated that STUB1 deficiency leads both to the intracellular accumulation of protein aggregates and to an increase in the secretion of small extracellular vesicles (sEVs), including exosomes. Secreted sEVs are enriched in ubiquitinated and/or undegraded proteins and protein oligomers. Data also indicates that oxidative stress induces an increase in the release of sEVs in cells depleted from STUB1. Overall, the results presented here suggest that cells use exosomes to dispose of damaged and/or undegraded proteins as a means to reduce intracellular accumulation of proteotoxic material. (hide) EV-METRIC 67% (94th 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 Microvesicles (MVs) 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: CD63/ GAPDH/ CANX/ Na/K A1 ATPase non-EV: Proteomics no EV density (g/ml) 1.15 Show all info Study aim Function/Biogenesis/cargo sorting Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells ARPE-19 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: time(min) 70 Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 120000 Wash: volume per pellet (ml) 38 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 16 Lowest density fraction 0.4M Highest density fraction 2.5M Total gradient volume, incl. sample (mL) 8 Sample volume (mL) 0.5 Orientation Bottom-up Rotor type Type 70.1Ti Speed (g) 210000 Duration (min) 960 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 38 Pelleting: duration (min) 70 Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 120000 EV-subtype Distinction between multiple subtypes Size Used subtypes >200 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD63/ GAPDH/ Na/K A1 ATPase/ CANX Characterization: Lipid analysis No EM EM-type Transmission-EM Image type Close-up

	EV190055	4/4	Homo sapiens	ARPE-19	DG (d)(U)C	Ferreira JV	2019	67%
Study summary Full title Exosomes and STUB1/CHIP cooperate to maintain intracellular proteostasis. All authors Ferreira JV, Rosa Soares A, Ramalho JS, Ribeiro-Rodrigues T, Máximo C, Zuzarte M, Girão H, Pereira P. Journal PLoS One Abstract Deregulation of proteostasis is a main feature of many age-related diseases, often leading to the ac (show more...)Deregulation of proteostasis is a main feature of many age-related diseases, often leading to the accumulation of toxic oligomers and insoluble protein aggregates that accumulate intracellularly or in the extracellular space. To understand the mechanisms whereby toxic or otherwise unwanted proteins are secreted to the extracellular space, we inactivated the quality-control and proteostasis regulator ubiquitin ligase STUB1/CHIP. Data indicated that STUB1 deficiency leads both to the intracellular accumulation of protein aggregates and to an increase in the secretion of small extracellular vesicles (sEVs), including exosomes. Secreted sEVs are enriched in ubiquitinated and/or undegraded proteins and protein oligomers. Data also indicates that oxidative stress induces an increase in the release of sEVs in cells depleted from STUB1. Overall, the results presented here suggest that cells use exosomes to dispose of damaged and/or undegraded proteins as a means to reduce intracellular accumulation of proteotoxic material. (hide) EV-METRIC 67% (94th 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 Microvesicles (MVs) 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: CD63/ GAPDH/ CANX/ Na/K A1 ATPase non-EV: Proteomics no EV density (g/ml) 1.15 Show all info Study aim Function/Biogenesis/cargo sorting Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells ARPE-19 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: time(min) 70 Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 120000 Wash: volume per pellet (ml) 38 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 16 Lowest density fraction 0.4M Highest density fraction 2.5M Total gradient volume, incl. sample (mL) 8 Sample volume (mL) 0.5 Orientation Bottom-up Rotor type Type 70.1Ti Speed (g) 210000 Duration (min) 960 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 38 Pelleting: duration (min) 70 Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 120000 EV-subtype Distinction between multiple subtypes Size Used subtypes >200 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins GAPDH/ Na/K A1 ATPase/ CANX/ CD63 Characterization: Lipid analysis No EM EM-type Transmission-EM Image type Close-up

	EV190040	7/12	Homo sapiens	MCF7	DG UF	Geeurickx E	2019	67%
Study summary Full title The generation and use of recombinant extracellular vesicles as biological reference material. All authors Geeurickx E, Tulkens J, Dhondt B, Van Deun J, Lippens L, Vergauwen G, Heyrman E, De Sutter D, Gevaert K, Impens F, Miinalainen I, Van Bockstal PJ, De Beer T, Wauben MHM, Nolte-'t-Hoen ENM, Bloch K, Swinnen JV, van der Pol E, Nieuwland R, Braems G, Callewaert N, Mestdagh P, Vandesompele J, Denys H, Eyckerman S, De Wever O, Hendrix A. Journal Nat Commun Abstract Recent years have seen an increase of extracellular vesicle (EV) research geared towards biological (show more...)Recent years have seen an increase of extracellular vesicle (EV) research geared towards biological understanding, diagnostics and therapy. However, EV data interpretation remains challenging owing to complexity of biofluids and technical variation introduced during sample preparation and analysis. To understand and mitigate these limitations, we generated trackable recombinant EV (rEV) as a biological reference material. Employing complementary characterization methods, we demonstrate that rEV are stable and bear physical and biochemical traits characteristic of sample EV. Furthermore, rEV can be quantified using fluorescence-, RNA- and protein-based technologies available in routine laboratories. Spiking rEV in biofluids allows recovery efficiencies of commonly implemented EV separation methods to be identified, intra-method and inter-user variability induced by sample handling to be defined, and to normalize and improve sensitivity of EV enumerations. We anticipate that rEV will aid EV-based sample preparation and analysis, data normalization, method development and instrument calibration in various research and biomedical applications. (hide) EV-METRIC 67% (94th 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 UF Protein markers EV: non-EV: Proteomics no EV density (g/ml) 1.076 1.088 g/ml Show all info Study aim New methodological development/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells MCF7 EV-harvesting Medium Serum free medium Cell viability (%) 96 Separation Method Density gradient Only used for validation of main results Yes Type Discontinuous Number of initial discontinuous layers 11 Lowest density fraction 6 Highest density fraction 18 Total gradient volume, incl. sample (mL) 15 Sample volume (mL) 1 Orientation Top-down Rotor type SW 32.1 Ti Speed (g) 186700 Duration (min) 116 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 16 Pelleting: duration (min) 180 Pelleting: rotor type SW 32.1 Ti Pelleting: speed (g) 100000 Ultra filtration Cut-off size (kDa) 10 Membrane type Regenerated cellulose EV-subtype Distinction between multiple subtypes Density Protein Concentration Method Fluorometric assay (e.g. Qubit, NanoOrange,...) Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Not Reported EV concentration Yes EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV190038	1/3	Homo sapiens	Blood plasma	(d)(U)C	García-Silva S	2019	67%
Study summary Full title Use of extracellular vesicles from lymphatic drainage as surrogate markers of melanoma progression and BRAF V600E mutation. All authors García-Silva S, Benito-Martín A, Sánchez-Redondo S, Hernández-Barranco A, Ximénez-Embún P, Nogués L, Mazariegos MS, Brinkmann K, Amor López A, Meyer L, Rodríguez C, García-Martín C, Boskovic J, Letón R, Montero C, Robledo M, Santambrogio L, Sue Brady M, Szumera-Ciećkiewicz A, Kalinowska I, Skog J, Noerholm M, Muñoz J, Ortiz-Romero PL, Ruano Y, Rodríguez-Peralto JL, Rutkowski P, Peinado H. Journal J Exp Med Abstract Liquid biopsies from cancer patients have the potential to improve diagnosis and prognosis. The asse (show more...)Liquid biopsies from cancer patients have the potential to improve diagnosis and prognosis. The assessment of surrogate markers of tumor progression in circulating extracellular vesicles could be a powerful non-invasive approach in this setting. We have characterized extracellular vesicles purified from the lymphatic drainage also known as exudative seroma (ES) of stage III melanoma patients obtained after lymphadenectomy. Proteomic analysis showed that seroma-derived exosomes are enriched in proteins resembling melanoma progression. In addition, we found that the BRAFV600E mutation can be detected in ES-derived extracellular vesicles and its detection correlated with patients at risk of relapse. (hide) EV-METRIC 67% (94th 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 Melanoma patients stage III 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 (d)(U)C Protein markers EV: CD63/ GADPH/ CD81/ HSP90/ TRP2/ CD9 non-EV: Proteomics yes Show all info Study aim 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 Type 50.4 Ti Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 3 Wash: time (min) 70 Wash: Rotor Type Type 50.4 Ti Wash: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? No Detected EV-associated proteins CD9/ CD63/ GADPH/ CD81/ TRP2 Not detected EV-associated proteins HSP90 Proteomics database ProteomeXchange Consortium Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Median Reported size (nm) 126 EV concentration Yes EM EM-type Transmission-EM Image type Close-up, Wide-field Report size (nm) 30.4

	EV190033	1/2	Homo sapiens	Blood plasma	(d)(U)C Filtration	Li Min	2019	67%
Study summary Full title Evaluation of circulating small extracellular vesicles derived miRNAs as biomarkers of early colon cancer: a comparison with plasma total miRNAs All authors Li Min, Shengtao Zhu, Lei Chen, Xiang Liu, Rui Wei, Libo Zhao, Yuqing Yang, Zheng Zhang, Guanyi Kong, Peng Li & Shutian Zhang Journal J Extracell Vesicles Abstract Early diagnosis of colon cancer (CC) is clinically important, as it can significantly improve patien (show more...)Early diagnosis of colon cancer (CC) is clinically important, as it can significantly improve patients’ survival rate and quality of life. Although the potential role for small extracellular vesicles (sEVs) in early detection of many diseases has been repeatedly mentioned, systematic screening of plasma sEVs derived early CC specific biomarkers has not yet been reported. In this work, plasma sEVs enriched fractions were derived from 15 early-stage (TisN0M0) CC patients and 10 normal controls (NC). RNA sequencing identified a total number of 95 sEVs enriched fraction derived miRNAs with differential expression between CC and NC, most of which (60/95) was in well accordance with tissue results in the Cancer Genome Atlas (TCGA) dataset. Among those miRNAs, we selected let-7b-3p, miR-139-3p, miR-145-3p, and miR-150-3p for further validation in an independent cohort consisting of 134 participants (58 CC and 76 NC). In the validation cohort, the AUC of 4 individual miRNAs ranged from 0.680 to 0.792. A logistic model combining two miRNAs (i.e. let-7b-3p and miR-145-3p) achieved an AUC of 0.901. Adding the 3rd miRNA into this model can further increase the AUC to 0.927. Side by side comparison revealed that sEVs miRNA profile outperformed cell-free plasma miRNA in the diagnosis of early CC. In conclusion, we suggested that circulating sEVs enriched fractions have a distinct miRNA profile in CC patients, and sEVs derived miRNA could be used as a promising biomarker to detect CC at an early stage. (hide) EV-METRIC 67% (94th 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 (d)(U)C Filtration Protein markers EV: TSG101/ Alix/ CD63 non-EV: Calnexin Proteomics no Show all info Study aim 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 Equal to or above 150,000 g Pelleting performed Yes Pelleting: time(min) 240 Pelleting: rotor type P50AT2-986 Pelleting: speed (g) 150000 Wash: volume per pellet (ml) 1 Wash: time (min) 120 Wash: Rotor Type P50A3-0099 Wash: speed (g) 150000 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD63/ TSG101/ Alix Not detected contaminants Calnexin Characterization: RNA analysis RNA analysis Type (RT)(q)PCR;RNA sequencing Database No Proteinase treatment Yes Moment of Proteinase treatment After Proteinase type Proteinase K Proteinase concentration 100 RNAse treatment Yes Moment of RNAse treatment After RNAse type RNase A RNAse concentration 0.01 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 75-200 EV concentration Yes EM EM-type Transmission-EM Image type Close-up, Wide-field Report size (nm)

	EV190024	2/10	Homo sapiens	Foreskin primary fibroblasts	(d)(U)C	Michela Borghesan	2019	67%
Study summary Full title Small Extracellular Vesicles Are Key Regulators of Non-cell Autonomous Intercellular Communication in Senescence via the Interferon Protein IFITM3 All authors Michela Borghesan, Juan Fafián-Labora, Olga Eleftheriadou, Paula Carpintero-Fernández, Marta Paez-Ribes, Gema Vizcay-Barrena, Avital Swisa, Dror Kolodkin-Gal, Pilar Ximénez-Embún, Robert Lowe, Belen Martín-Martín, Hector Peinado, Javier Muñoz, Roland A. Fleck, Yuval Dor, Ittai Ben-Porath, Anna Vossenkamper, Daniel Muñoz-Espin and Ana O’Loghlen Journal Cell Rep Abstract Senescence is a cellular phenotype present in health and disease, characterized by a stable cell-cyc (show more...)Senescence is a cellular phenotype present in health and disease, characterized by a stable cell-cycle arrest and an inflammatory response called senescence-associated secretory phenotype (SASP). The SASP is important in influencing the behavior of neighboring cells and altering the microenvironment; yet, this role has been mainly attributed to soluble factors. Here, we show that both the soluble factors and small extracellular vesicles (sEVs) are capable of transmitting paracrine senescence to nearby cells. Analysis of individual cells internalizing sEVs, using a Cre-reporter system, show a positive correlation between sEV uptake and senescence activation. We find an increase in the number of multivesicular bodies during senescence in vivo. sEV protein characterization by mass spectrometry (MS) followed by a functional siRNA screen identify interferon-induced transmembrane protein 3 (IFITM3) as being partially responsible for transmitting senescence to normal cells. We find that sEVs contribute to paracrine senescence. (hide) EV-METRIC 67% (94th 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 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 (d)(U)C Protein markers EV: CD81/ IFITM3/ CD63/ CD9/ Alix non-EV: calnexin/ Cox IV Proteomics yes Show all info Study aim Function/Biomarker/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Foreskin primary fibroblasts EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g 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 50,000 g and 100,000 g Pelleting performed Yes Pelleting: time(min) 80 Pelleting: rotor type T-865 Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 15 Wash: time (min) 80 Wash: Rotor Type T-865 Wash: speed (g) 100000 EV-subtype Distinction between multiple subtypes Size Used subtypes Small vesicles (below 200nm) Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Antibody details provided? No Detected EV-associated proteins Alix/ CD63 Not detected EV-associated proteins CD81/ CD9 Not detected contaminants Cox IV/ calnexin Flow cytometry specific beads Antibody details provided? No Antibody dilution provided? No Detected EV-associated proteins IFITM3/ CD63/ CD81 Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 150 EV concentration Yes Particle analysis: flow cytometry Flow cytometer type NovoCyte Flow Cytometer Hardware adjustment Calibration bead size 4 Report type Not Reported EM EM-type Transmission-EM Image type Wide-field

	EV190024	4/10	Homo sapiens	Foreskin primary fibroblasts	(d)(U)C	Michela Borghesan	2019	67%
Study summary Full title Small Extracellular Vesicles Are Key Regulators of Non-cell Autonomous Intercellular Communication in Senescence via the Interferon Protein IFITM3 All authors Michela Borghesan, Juan Fafián-Labora, Olga Eleftheriadou, Paula Carpintero-Fernández, Marta Paez-Ribes, Gema Vizcay-Barrena, Avital Swisa, Dror Kolodkin-Gal, Pilar Ximénez-Embún, Robert Lowe, Belen Martín-Martín, Hector Peinado, Javier Muñoz, Roland A. Fleck, Yuval Dor, Ittai Ben-Porath, Anna Vossenkamper, Daniel Muñoz-Espin and Ana O’Loghlen Journal Cell Rep Abstract Senescence is a cellular phenotype present in health and disease, characterized by a stable cell-cyc (show more...)Senescence is a cellular phenotype present in health and disease, characterized by a stable cell-cycle arrest and an inflammatory response called senescence-associated secretory phenotype (SASP). The SASP is important in influencing the behavior of neighboring cells and altering the microenvironment; yet, this role has been mainly attributed to soluble factors. Here, we show that both the soluble factors and small extracellular vesicles (sEVs) are capable of transmitting paracrine senescence to nearby cells. Analysis of individual cells internalizing sEVs, using a Cre-reporter system, show a positive correlation between sEV uptake and senescence activation. We find an increase in the number of multivesicular bodies during senescence in vivo. sEV protein characterization by mass spectrometry (MS) followed by a functional siRNA screen identify interferon-induced transmembrane protein 3 (IFITM3) as being partially responsible for transmitting senescence to normal cells. We find that sEVs contribute to paracrine senescence. (hide) EV-METRIC 67% (94th 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 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 (d)(U)C Protein markers EV: CD81/ Alix/ TSG101/ CD63/ CD9 non-EV: calnexin/ Cox IV Proteomics yes Show all info Study aim Function/Biomarker/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Foreskin primary fibroblasts EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g 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 50,000 g and 100,000 g Pelleting performed Yes Pelleting: time(min) 80 Pelleting: rotor type T-865 Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 15 Wash: time (min) 80 Wash: Rotor Type T-865 Wash: speed (g) 100000 EV-subtype Distinction between multiple subtypes Size Used subtypes Small vesicles (below 200nm) Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Antibody details provided? No Detected EV-associated proteins Alix/ CD63/ TSG101 Not detected EV-associated proteins CD81/ CD9 Not detected contaminants Cox IV/ calnexin Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) below 200 EV concentration Yes Particle analysis: flow cytometry Flow cytometer type NovoCyte Flow Cytometer Hardware adjustment Calibration bead size 4 Report type Not Reported EM EM-type Transmission-EM Image type Wide-field

	EV190024	6/10	Homo sapiens	Foreskin primary fibroblasts	(d)(U)C	Michela Borghesan	2019	67%
Study summary Full title Small Extracellular Vesicles Are Key Regulators of Non-cell Autonomous Intercellular Communication in Senescence via the Interferon Protein IFITM3 All authors Michela Borghesan, Juan Fafián-Labora, Olga Eleftheriadou, Paula Carpintero-Fernández, Marta Paez-Ribes, Gema Vizcay-Barrena, Avital Swisa, Dror Kolodkin-Gal, Pilar Ximénez-Embún, Robert Lowe, Belen Martín-Martín, Hector Peinado, Javier Muñoz, Roland A. Fleck, Yuval Dor, Ittai Ben-Porath, Anna Vossenkamper, Daniel Muñoz-Espin and Ana O’Loghlen Journal Cell Rep Abstract Senescence is a cellular phenotype present in health and disease, characterized by a stable cell-cyc (show more...)Senescence is a cellular phenotype present in health and disease, characterized by a stable cell-cycle arrest and an inflammatory response called senescence-associated secretory phenotype (SASP). The SASP is important in influencing the behavior of neighboring cells and altering the microenvironment; yet, this role has been mainly attributed to soluble factors. Here, we show that both the soluble factors and small extracellular vesicles (sEVs) are capable of transmitting paracrine senescence to nearby cells. Analysis of individual cells internalizing sEVs, using a Cre-reporter system, show a positive correlation between sEV uptake and senescence activation. We find an increase in the number of multivesicular bodies during senescence in vivo. sEV protein characterization by mass spectrometry (MS) followed by a functional siRNA screen identify interferon-induced transmembrane protein 3 (IFITM3) as being partially responsible for transmitting senescence to normal cells. We find that sEVs contribute to paracrine senescence. (hide) EV-METRIC 67% (94th 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 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 (d)(U)C Protein markers EV: IFITM3/ TSG101/ CD63/ CD81/ Alix/ CD9 non-EV: calnexin/ Cox IV Proteomics yes Show all info Study aim Function/Biomarker/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Foreskin primary fibroblasts EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g 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 50,000 g and 100,000 g Pelleting performed Yes Pelleting: time(min) 80 Pelleting: rotor type T-865 Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 15 Wash: time (min) 80 Wash: Rotor Type T-865 Wash: speed (g) 100000 EV-subtype Distinction between multiple subtypes Size Used subtypes Small vesicles (below 200nm) Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Antibody details provided? No Detected EV-associated proteins Alix/ TSG101/ CD63 Not detected EV-associated proteins CD81/ CD9 Not detected contaminants Cox IV/ calnexin Flow cytometry specific beads Antibody details provided? No Antibody dilution provided? No Detected EV-associated proteins IFITM3/ CD63/ CD81 Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) below 200 EV concentration Yes Particle analysis: flow cytometry Flow cytometer type NovoCyte Flow Cytometer Hardware adjustment Calibration bead size 4 EM EM-type Transmission-EM Image type Wide-field

	EV190024	8/10	Homo sapiens	Foreskin primary fibroblasts	(d)(U)C	Michela Borghesan	2019	67%
Study summary Full title Small Extracellular Vesicles Are Key Regulators of Non-cell Autonomous Intercellular Communication in Senescence via the Interferon Protein IFITM3 All authors Michela Borghesan, Juan Fafián-Labora, Olga Eleftheriadou, Paula Carpintero-Fernández, Marta Paez-Ribes, Gema Vizcay-Barrena, Avital Swisa, Dror Kolodkin-Gal, Pilar Ximénez-Embún, Robert Lowe, Belen Martín-Martín, Hector Peinado, Javier Muñoz, Roland A. Fleck, Yuval Dor, Ittai Ben-Porath, Anna Vossenkamper, Daniel Muñoz-Espin and Ana O’Loghlen Journal Cell Rep Abstract Senescence is a cellular phenotype present in health and disease, characterized by a stable cell-cyc (show more...)Senescence is a cellular phenotype present in health and disease, characterized by a stable cell-cycle arrest and an inflammatory response called senescence-associated secretory phenotype (SASP). The SASP is important in influencing the behavior of neighboring cells and altering the microenvironment; yet, this role has been mainly attributed to soluble factors. Here, we show that both the soluble factors and small extracellular vesicles (sEVs) are capable of transmitting paracrine senescence to nearby cells. Analysis of individual cells internalizing sEVs, using a Cre-reporter system, show a positive correlation between sEV uptake and senescence activation. We find an increase in the number of multivesicular bodies during senescence in vivo. sEV protein characterization by mass spectrometry (MS) followed by a functional siRNA screen identify interferon-induced transmembrane protein 3 (IFITM3) as being partially responsible for transmitting senescence to normal cells. We find that sEVs contribute to paracrine senescence. (hide) EV-METRIC 67% (94th 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 inducible H-RASG12V 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 (d)(U)C Protein markers EV: IFITM3/ TSG101/ CD63/ CD81/ Alix/ CD9 non-EV: calnexin/ Cox IV Proteomics yes Show all info Study aim Function/Biomarker/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Foreskin primary fibroblasts EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g 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 50,000 g and 100,000 g Pelleting performed Yes Pelleting: time(min) 80 Pelleting: rotor type T-865 Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 15 Wash: time (min) 80 Wash: Rotor Type T-865 Wash: speed (g) 100000 EV-subtype Distinction between multiple subtypes Size Used subtypes Small vesicles (below 200nm) Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Antibody details provided? No Detected EV-associated proteins Alix/ TSG101/ CD63 Not detected EV-associated proteins CD81/ CD9 Not detected contaminants Cox IV/ calnexin Flow cytometry specific beads Antibody details provided? No Antibody dilution provided? No Detected EV-associated proteins IFITM3/ CD63/ CD81 Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) below 200 EV concentration Yes Particle analysis: flow cytometry Flow cytometer type NovoCyte Flow Cytometer Hardware adjustment Calibration bead size 4 EM EM-type Transmission-EM Image type Wide-field

	EV190024	10/10	Homo sapiens	Foreskin primary fibroblasts	(d)(U)C	Michela Borghesan	2019	67%
Study summary Full title Small Extracellular Vesicles Are Key Regulators of Non-cell Autonomous Intercellular Communication in Senescence via the Interferon Protein IFITM3 All authors Michela Borghesan, Juan Fafián-Labora, Olga Eleftheriadou, Paula Carpintero-Fernández, Marta Paez-Ribes, Gema Vizcay-Barrena, Avital Swisa, Dror Kolodkin-Gal, Pilar Ximénez-Embún, Robert Lowe, Belen Martín-Martín, Hector Peinado, Javier Muñoz, Roland A. Fleck, Yuval Dor, Ittai Ben-Porath, Anna Vossenkamper, Daniel Muñoz-Espin and Ana O’Loghlen Journal Cell Rep Abstract Senescence is a cellular phenotype present in health and disease, characterized by a stable cell-cyc (show more...)Senescence is a cellular phenotype present in health and disease, characterized by a stable cell-cycle arrest and an inflammatory response called senescence-associated secretory phenotype (SASP). The SASP is important in influencing the behavior of neighboring cells and altering the microenvironment; yet, this role has been mainly attributed to soluble factors. Here, we show that both the soluble factors and small extracellular vesicles (sEVs) are capable of transmitting paracrine senescence to nearby cells. Analysis of individual cells internalizing sEVs, using a Cre-reporter system, show a positive correlation between sEV uptake and senescence activation. We find an increase in the number of multivesicular bodies during senescence in vivo. sEV protein characterization by mass spectrometry (MS) followed by a functional siRNA screen identify interferon-induced transmembrane protein 3 (IFITM3) as being partially responsible for transmitting senescence to normal cells. We find that sEVs contribute to paracrine senescence. (hide) EV-METRIC 67% (94th 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 inducible H-RASG12V Focus vesicles small extracellular vesicles / Other 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: CD81/ Alix/ TSG101/ CD63/ CD9 non-EV: calnexin/ Cox IV Proteomics yes Show all info Study aim Function/Biomarker/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Foreskin primary fibroblasts EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g 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 50,000 g and 100,000 g Pelleting performed Yes Pelleting: time(min) 80 Pelleting: rotor type T-865 Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 15 Wash: time (min) 80 Wash: Rotor Type T-865 Wash: speed (g) 100000 EV-subtype Distinction between multiple subtypes Size Used subtypes Small vesicles (below 200nm) Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Antibody details provided? No Detected EV-associated proteins Alix/ TSG101/ CD63 Not detected EV-associated proteins CD9/ CD81 Not detected contaminants Cox IV/ calnexin Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) below 200 EV concentration Yes Particle analysis: flow cytometry Flow cytometer type NovoCyte Flow Cytometer Hardware adjustment Calibration bead size 4 Report type Not Reported EM EM-type Transmission-EM Image type Wide-field

	EV180050	1/6	Homo sapiens	liver stem cells	(d)(U)C Filtration	Alice Gualerzi	2019	66%
Study summary Full title Raman spectroscopy as a quick tool to assess purity of extracellular vesicle preparations and predict their functionality All authors Alice Gualerzi, Sander Alexander Antonius Kooijmans, Stefania Niada, Silvia Picciolini, Anna Teresa Brini, Giovanni Camussi & Marzia Bedoni Journal J Extracell Vesicles Abstract Extracellular vesicles (EVs) from a variety of stem cell sources are believed to harbour regenerativ (show more...)Extracellular vesicles (EVs) from a variety of stem cell sources are believed to harbour regenerative capacity, which may be exploited for therapeutic purposes. Because of EV interaction with other soluble secreted factors, EV activity may depend on the employed purification method, which limits cross-study comparisons and therapeutic development. Raman spectroscopy (RS) is a quick and easy method to assess EV purity and composition, giving in-depth biochemical overview on EV preparation. Hereby, we show how this method can be used to characterise EVs isolated from human liver stem cells and bone marrow mesenchymal stem/stromal cells by means of conventional ultracentrifugation (UC) and size exclusion chromatography (SEC) protocols. The obtained EV preparations were demonstrated to be characterised by different degrees of purity and a specific Raman fingerprint that represents both the cell source and the isolation procedure used. Moreover, RS provided useful hints to explore the factors underlying the functional diversity of EV preparations from the same cell source, thus representing a valuable tool to assess EV quality prior to functional assays or therapeutic application. (hide) EV-METRIC 66% (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 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 Adj. k-factor 156.9 (pelleting) / 156.9 (washing) Protein markers EV: TSG101/ CD63/ CD81/ Alix/ beta-actin/ Flotillin-1/ CD9 non-EV: Calnexin/ Calreticulin Proteomics no Show all info Study aim New methodological development, Technical analysis comparing/optimizing EV-related methods, Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells liver stem cells 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 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 70 Pelleting: rotor type Type 70 Ti Pelleting: speed (g) 100000 Pelleting: adjusted k-factor 156.9 Wash: time (min) 70 Wash: Rotor Type Type 70 Ti Wash: speed (g) 100000 Wash: adjusted k-factor 156.9 Filtration steps > 0.45 µm, Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins Alix, CD63, CD81, CD9, Flotillin-1, TSG101, beta-actin Not detected contaminants Calnexin, Calreticulin Characterization: Lipid analysis No Characterization: Particle analysis PMID previous EV particle analysis Other Extra particle analysis NTA Report type Mean Reported size (nm) 189 ± 27 EV concentration Yes EM EM-type Transmission-EM Image type Wide-field Other particle analysis name(1) Raman spectroscopy

	EV180050	2/6	Homo sapiens	bone marrow-derived mesenchymal stem cells	(d)(U)C Filtration	Alice Gualerzi	2019	66%
Study summary Full title Raman spectroscopy as a quick tool to assess purity of extracellular vesicle preparations and predict their functionality All authors Alice Gualerzi, Sander Alexander Antonius Kooijmans, Stefania Niada, Silvia Picciolini, Anna Teresa Brini, Giovanni Camussi & Marzia Bedoni Journal J Extracell Vesicles Abstract Extracellular vesicles (EVs) from a variety of stem cell sources are believed to harbour regenerativ (show more...)Extracellular vesicles (EVs) from a variety of stem cell sources are believed to harbour regenerative capacity, which may be exploited for therapeutic purposes. Because of EV interaction with other soluble secreted factors, EV activity may depend on the employed purification method, which limits cross-study comparisons and therapeutic development. Raman spectroscopy (RS) is a quick and easy method to assess EV purity and composition, giving in-depth biochemical overview on EV preparation. Hereby, we show how this method can be used to characterise EVs isolated from human liver stem cells and bone marrow mesenchymal stem/stromal cells by means of conventional ultracentrifugation (UC) and size exclusion chromatography (SEC) protocols. The obtained EV preparations were demonstrated to be characterised by different degrees of purity and a specific Raman fingerprint that represents both the cell source and the isolation procedure used. Moreover, RS provided useful hints to explore the factors underlying the functional diversity of EV preparations from the same cell source, thus representing a valuable tool to assess EV quality prior to functional assays or therapeutic application. (hide) EV-METRIC 66% (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 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 Adj. k-factor 156.9 (pelleting) Protein markers EV: CD81/ Flotillin-1/ CD63 non-EV: Calnexin/ Calreticulin Proteomics no Show all info Study aim New methodological development, Technical analysis comparing/optimizing EV-related methods, Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells bone marrow-derived mesenchymal stem cells 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 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 70 Pelleting: rotor type Type 70 Ti Pelleting: speed (g) 100000 Pelleting: adjusted k-factor 156.9 Filtration steps > 0.45 µm, Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD63, CD81, Flotillin-1 Not detected contaminants Calnexin, Calreticulin Characterization: Lipid analysis No Characterization: Particle analysis PMID previous EV particle analysis Other Extra particle analysis NTA Report type Mean Reported size (nm) 212 ± 34 EV concentration Yes EM EM-type Transmission-EM Image type Wide-field Other particle analysis name(1) Raman spectroscopy

	EV180050	5/6	Homo sapiens	bone marrow-derived mesenchymal stem cells	(d)(U)C Filtration	Alice Gualerzi	2019	66%
Study summary Full title Raman spectroscopy as a quick tool to assess purity of extracellular vesicle preparations and predict their functionality All authors Alice Gualerzi, Sander Alexander Antonius Kooijmans, Stefania Niada, Silvia Picciolini, Anna Teresa Brini, Giovanni Camussi & Marzia Bedoni Journal J Extracell Vesicles Abstract Extracellular vesicles (EVs) from a variety of stem cell sources are believed to harbour regenerativ (show more...)Extracellular vesicles (EVs) from a variety of stem cell sources are believed to harbour regenerative capacity, which may be exploited for therapeutic purposes. Because of EV interaction with other soluble secreted factors, EV activity may depend on the employed purification method, which limits cross-study comparisons and therapeutic development. Raman spectroscopy (RS) is a quick and easy method to assess EV purity and composition, giving in-depth biochemical overview on EV preparation. Hereby, we show how this method can be used to characterise EVs isolated from human liver stem cells and bone marrow mesenchymal stem/stromal cells by means of conventional ultracentrifugation (UC) and size exclusion chromatography (SEC) protocols. The obtained EV preparations were demonstrated to be characterised by different degrees of purity and a specific Raman fingerprint that represents both the cell source and the isolation procedure used. Moreover, RS provided useful hints to explore the factors underlying the functional diversity of EV preparations from the same cell source, thus representing a valuable tool to assess EV quality prior to functional assays or therapeutic application. (hide) EV-METRIC 66% (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 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 Adj. k-factor 156.9 (pelleting) / 156.9 (washing) Protein markers EV: CD81/ Flotillin-1/ CD63 non-EV: Calnexin/ Calreticulin Proteomics no Show all info Study aim New methodological development, Technical analysis comparing/optimizing EV-related methods, Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells bone marrow-derived mesenchymal stem cells 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 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 70 Pelleting: rotor type Type 70 Ti Pelleting: speed (g) 100000 Pelleting: adjusted k-factor 156.9 Wash: time (min) 70 Wash: Rotor Type Type 70 Ti Wash: speed (g) 100000 Wash: adjusted k-factor 156.9 Filtration steps > 0.45 µm, Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD63, CD81, Flotillin-1 Not detected contaminants Calnexin, Calreticulin Characterization: Lipid analysis No Characterization: Particle analysis PMID previous EV particle analysis Other Extra particle analysis NTA Report type Mean Reported size (nm) 204 ± 42 EV concentration Yes EM EM-type Transmission-EM Image type Wide-field Other particle analysis name(1) Raman spectroscopy

	EV180050	6/6	Homo sapiens	liver stem cells	(d)(U)C Filtration	Alice Gualerzi	2019	66%
Study summary Full title Raman spectroscopy as a quick tool to assess purity of extracellular vesicle preparations and predict their functionality All authors Alice Gualerzi, Sander Alexander Antonius Kooijmans, Stefania Niada, Silvia Picciolini, Anna Teresa Brini, Giovanni Camussi & Marzia Bedoni Journal J Extracell Vesicles Abstract Extracellular vesicles (EVs) from a variety of stem cell sources are believed to harbour regenerativ (show more...)Extracellular vesicles (EVs) from a variety of stem cell sources are believed to harbour regenerative capacity, which may be exploited for therapeutic purposes. Because of EV interaction with other soluble secreted factors, EV activity may depend on the employed purification method, which limits cross-study comparisons and therapeutic development. Raman spectroscopy (RS) is a quick and easy method to assess EV purity and composition, giving in-depth biochemical overview on EV preparation. Hereby, we show how this method can be used to characterise EVs isolated from human liver stem cells and bone marrow mesenchymal stem/stromal cells by means of conventional ultracentrifugation (UC) and size exclusion chromatography (SEC) protocols. The obtained EV preparations were demonstrated to be characterised by different degrees of purity and a specific Raman fingerprint that represents both the cell source and the isolation procedure used. Moreover, RS provided useful hints to explore the factors underlying the functional diversity of EV preparations from the same cell source, thus representing a valuable tool to assess EV quality prior to functional assays or therapeutic application. (hide) EV-METRIC 66% (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 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 Adj. k-factor 156.9 (pelleting) Protein markers EV: TSG101/ CD63/ CD81/ Alix/ beta-actin/ Flotillin-1/ CD9 non-EV: Calnexin/ Calreticulin Proteomics no Show all info Study aim New methodological development, Technical analysis comparing/optimizing EV-related methods, Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells liver stem cells 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 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 70 Pelleting: rotor type Type 70 Ti Pelleting: speed (g) 100000 Pelleting: adjusted k-factor 156.9 Filtration steps > 0.45 µm, Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins Alix, CD63, CD81, CD9, Flotillin-1, TSG101, beta-actin Not detected contaminants Calnexin, Calreticulin Characterization: Lipid analysis No Characterization: Particle analysis PMID previous EV particle analysis Other Extra particle analysis NTA Report type Mean Reported size (nm) 184 ± 33 EV concentration Yes EM EM-type Transmission-EM Image type Wide-field Other particle analysis name(1) Raman spectroscopy

	EV180029	1/8	Homo sapiens	PC3	(d)(U)C	Palviainen, Mari	2019	66%
Study summary Full title Metabolic signature of extracellular vesicles depends on the cell culture conditions All authors Mari Palviainen ORCID Icon, Heikki Saari ORCID Icon, Olli Kärkkäinen ORCID Icon, Jenna Pekkinen, Seppo Auriola, Marjo Yliperttula, Maija Puhka, Kati Hanhineva & Pia R.-M. Siljander Journal J Extracell Vesicles Abstract One of the greatest bottlenecks in extracellular vesicle (EV) research is the production of sufficie (show more...)One of the greatest bottlenecks in extracellular vesicle (EV) research is the production of sufficient material in a consistent and effective way using in vitro cell models. Although the production of EVs in bioreactors maximizes EV yield in comparison to conventional cell cultures, the impact of their cell growth conditions on EVs has not yet been established. In this study, we grew two prostate cancer cell lines, PC-3 and VCaP, in conventional cell culture dishes and in two-chamber bioreactors to elucidate how the growth environment affects the EV characteristics. Specifically, we wanted to investigate the growth condition-dependent differences by non-targeted metabolite profiling using liquid chromatography–mass spectrometry (LC–MS) analysis. EVs were also characterized by their morphology, size distribution, and EV protein marker expression, and the EV yields were quantified by NTA. The use of bioreactor increased the EV yield >100 times compared to the conventional cell culture system. Regarding morphology, size distribution and surface markers, only minor differences were observed between the bioreactor-derived EVs (BR-EVs) and the EVs obtained from cells grown in conventional cell cultures (C-EVs). In contrast, metabolomic analysis revealed statistically significant differences in both polar and non-polar metabolites when the BR-EVs were compared to the C-EVs. The results show that the growth conditions markedly affected the EV metabolite profiles and that metabolomics was a sensitive tool to study molecular differences of EVs. We conclude that the cell culture conditions of EV production should be standardized and carefully detailed in publications and care should be taken when EVs from different production platforms are compared with each other for systemic effects. (hide) EV-METRIC 66% (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 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 Adj. k-factor 142.9 (pelleting) / 89.2 (washing) Protein markers EV: CD81/ TSG101/ CD29/ CD9 non-EV: calnexin Proteomics no Show all info Study aim Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells PC3 EV-harvesting Medium EV-depleted serum Preparation of EDS overnight (16h) at >=100,000g 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) 120 Pelleting: rotor type Type 50.2 Ti Pelleting: speed (g) 110000 Pelleting: adjusted k-factor 142.9 Wash: time (min) 120 Wash: Rotor Type TLA-55 Wash: speed (g) 100000 Wash: adjusted k-factor 89.20 Characterization: Protein analysis Protein Concentration Method Lowry-based assay Western Blot Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD9, CD81, TSG101, CD29 Not detected contaminants calnexin Characterization: Lipid analysis No Characterization: Particle analysis PMID previous EV particle analysis Electron microscopy Extra particle analysis NTA Report type Mean Reported size (nm) 144.8 EV concentration Yes EM EM-type Transmission-EM Image type Wide-field

	EV180029	2/8	Homo sapiens	VCaP	(d)(U)C	Palviainen, Mari	2019	66%
Study summary Full title Metabolic signature of extracellular vesicles depends on the cell culture conditions All authors Mari Palviainen ORCID Icon, Heikki Saari ORCID Icon, Olli Kärkkäinen ORCID Icon, Jenna Pekkinen, Seppo Auriola, Marjo Yliperttula, Maija Puhka, Kati Hanhineva & Pia R.-M. Siljander Journal J Extracell Vesicles Abstract One of the greatest bottlenecks in extracellular vesicle (EV) research is the production of sufficie (show more...)One of the greatest bottlenecks in extracellular vesicle (EV) research is the production of sufficient material in a consistent and effective way using in vitro cell models. Although the production of EVs in bioreactors maximizes EV yield in comparison to conventional cell cultures, the impact of their cell growth conditions on EVs has not yet been established. In this study, we grew two prostate cancer cell lines, PC-3 and VCaP, in conventional cell culture dishes and in two-chamber bioreactors to elucidate how the growth environment affects the EV characteristics. Specifically, we wanted to investigate the growth condition-dependent differences by non-targeted metabolite profiling using liquid chromatography–mass spectrometry (LC–MS) analysis. EVs were also characterized by their morphology, size distribution, and EV protein marker expression, and the EV yields were quantified by NTA. The use of bioreactor increased the EV yield >100 times compared to the conventional cell culture system. Regarding morphology, size distribution and surface markers, only minor differences were observed between the bioreactor-derived EVs (BR-EVs) and the EVs obtained from cells grown in conventional cell cultures (C-EVs). In contrast, metabolomic analysis revealed statistically significant differences in both polar and non-polar metabolites when the BR-EVs were compared to the C-EVs. The results show that the growth conditions markedly affected the EV metabolite profiles and that metabolomics was a sensitive tool to study molecular differences of EVs. We conclude that the cell culture conditions of EV production should be standardized and carefully detailed in publications and care should be taken when EVs from different production platforms are compared with each other for systemic effects. (hide) EV-METRIC 66% (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 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 Adj. k-factor 142.9 (pelleting) / 89.2 (washing) Protein markers EV: CD81/ TSG101/ CD29/ CD9 non-EV: calnexin Proteomics no Show all info Study aim Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells VCaP EV-harvesting Medium Serum-containing, but physical separation of serum EVs and secreted EVs (e.g. Bioreactor flask) 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) 120 Pelleting: rotor type Type 50.2 Ti Pelleting: speed (g) 110000 Pelleting: adjusted k-factor 142.9 Wash: time (min) 120 Wash: Rotor Type TLA-55 Wash: speed (g) 100000 Wash: adjusted k-factor 89.20 Characterization: Protein analysis Protein Concentration Method Lowry-based assay Western Blot Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD9, CD81, TSG101, CD29 Not detected contaminants calnexin Characterization: Lipid analysis No Characterization: Particle analysis PMID previous EV particle analysis Electron microscopy Extra particle analysis NTA Report type Mean Reported size (nm) 88 EV concentration Yes EM EM-type Transmission-EM Image type Close-up

	EV180029	3/8	Homo sapiens	PC3	(d)(U)C	Palviainen, Mari	2019	66%
Study summary Full title Metabolic signature of extracellular vesicles depends on the cell culture conditions All authors Mari Palviainen ORCID Icon, Heikki Saari ORCID Icon, Olli Kärkkäinen ORCID Icon, Jenna Pekkinen, Seppo Auriola, Marjo Yliperttula, Maija Puhka, Kati Hanhineva & Pia R.-M. Siljander Journal J Extracell Vesicles Abstract One of the greatest bottlenecks in extracellular vesicle (EV) research is the production of sufficie (show more...)One of the greatest bottlenecks in extracellular vesicle (EV) research is the production of sufficient material in a consistent and effective way using in vitro cell models. Although the production of EVs in bioreactors maximizes EV yield in comparison to conventional cell cultures, the impact of their cell growth conditions on EVs has not yet been established. In this study, we grew two prostate cancer cell lines, PC-3 and VCaP, in conventional cell culture dishes and in two-chamber bioreactors to elucidate how the growth environment affects the EV characteristics. Specifically, we wanted to investigate the growth condition-dependent differences by non-targeted metabolite profiling using liquid chromatography–mass spectrometry (LC–MS) analysis. EVs were also characterized by their morphology, size distribution, and EV protein marker expression, and the EV yields were quantified by NTA. The use of bioreactor increased the EV yield >100 times compared to the conventional cell culture system. Regarding morphology, size distribution and surface markers, only minor differences were observed between the bioreactor-derived EVs (BR-EVs) and the EVs obtained from cells grown in conventional cell cultures (C-EVs). In contrast, metabolomic analysis revealed statistically significant differences in both polar and non-polar metabolites when the BR-EVs were compared to the C-EVs. The results show that the growth conditions markedly affected the EV metabolite profiles and that metabolomics was a sensitive tool to study molecular differences of EVs. We conclude that the cell culture conditions of EV production should be standardized and carefully detailed in publications and care should be taken when EVs from different production platforms are compared with each other for systemic effects. (hide) EV-METRIC 66% (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 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 Adj. k-factor 785.9 (pelleting) / 89.2 (washing) Protein markers EV: CD81/ TSG101/ CD29/ CD9 non-EV: calnexin Proteomics no Show all info Study aim Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells PC3 EV-harvesting Medium EV-depleted serum Preparation of EDS overnight (16h) at >=100,000g 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 Yes Pelleting: time(min) 120 Pelleting: rotor type Type 50.2 Ti Pelleting: speed (g) 20000 Pelleting: adjusted k-factor 785.9 Wash: time (min) 120 Wash: Rotor Type TLA-55 Wash: speed (g) 100000 Wash: adjusted k-factor 89.20 Characterization: Protein analysis Protein Concentration Method Lowry-based assay Western Blot Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD9, CD81, TSG101, CD29 Not detected contaminants calnexin Characterization: Lipid analysis No Characterization: Particle analysis PMID previous EV particle analysis Electron microscopy Extra particle analysis NTA Report type Mean Reported size (nm) 178.3 EV concentration Yes EM EM-type Transmission-EM Image type Wide-field

	EV180029	4/8	Homo sapiens	VCaP	(d)(U)C	Palviainen, Mari	2019	66%
Study summary Full title Metabolic signature of extracellular vesicles depends on the cell culture conditions All authors Mari Palviainen ORCID Icon, Heikki Saari ORCID Icon, Olli Kärkkäinen ORCID Icon, Jenna Pekkinen, Seppo Auriola, Marjo Yliperttula, Maija Puhka, Kati Hanhineva & Pia R.-M. Siljander Journal J Extracell Vesicles Abstract One of the greatest bottlenecks in extracellular vesicle (EV) research is the production of sufficie (show more...)One of the greatest bottlenecks in extracellular vesicle (EV) research is the production of sufficient material in a consistent and effective way using in vitro cell models. Although the production of EVs in bioreactors maximizes EV yield in comparison to conventional cell cultures, the impact of their cell growth conditions on EVs has not yet been established. In this study, we grew two prostate cancer cell lines, PC-3 and VCaP, in conventional cell culture dishes and in two-chamber bioreactors to elucidate how the growth environment affects the EV characteristics. Specifically, we wanted to investigate the growth condition-dependent differences by non-targeted metabolite profiling using liquid chromatography–mass spectrometry (LC–MS) analysis. EVs were also characterized by their morphology, size distribution, and EV protein marker expression, and the EV yields were quantified by NTA. The use of bioreactor increased the EV yield >100 times compared to the conventional cell culture system. Regarding morphology, size distribution and surface markers, only minor differences were observed between the bioreactor-derived EVs (BR-EVs) and the EVs obtained from cells grown in conventional cell cultures (C-EVs). In contrast, metabolomic analysis revealed statistically significant differences in both polar and non-polar metabolites when the BR-EVs were compared to the C-EVs. The results show that the growth conditions markedly affected the EV metabolite profiles and that metabolomics was a sensitive tool to study molecular differences of EVs. We conclude that the cell culture conditions of EV production should be standardized and carefully detailed in publications and care should be taken when EVs from different production platforms are compared with each other for systemic effects. (hide) EV-METRIC 66% (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 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 Adj. k-factor 142.9 (pelleting) / 89.2 (washing) Protein markers EV: CD81/ TSG101/ CD29/ CD9 non-EV: calnexin Proteomics no Show all info Study aim Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells VCaP EV-harvesting Medium EV-depleted serum Preparation of EDS overnight (16h) at >=100,000g 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) 120 Pelleting: rotor type Type 50.2 Ti Pelleting: speed (g) 110000 Pelleting: adjusted k-factor 142.9 Wash: time (min) 120 Wash: Rotor Type TLA-55 Wash: speed (g) 100000 Wash: adjusted k-factor 89.20 Characterization: Protein analysis Protein Concentration Method Lowry-based assay Western Blot Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD9, CD81, TSG101, CD29 Not detected contaminants calnexin Characterization: Lipid analysis No Characterization: Particle analysis PMID previous EV particle analysis Electron microscopy Extra particle analysis NTA Report type Mean Reported size (nm) 111 EV concentration Yes EM EM-type Transmission-EM Image type Wide-field

	EV180029	5/8	Homo sapiens	VCaP	(d)(U)C	Palviainen, Mari	2019	66%
Study summary Full title Metabolic signature of extracellular vesicles depends on the cell culture conditions All authors Mari Palviainen ORCID Icon, Heikki Saari ORCID Icon, Olli Kärkkäinen ORCID Icon, Jenna Pekkinen, Seppo Auriola, Marjo Yliperttula, Maija Puhka, Kati Hanhineva & Pia R.-M. Siljander Journal J Extracell Vesicles Abstract One of the greatest bottlenecks in extracellular vesicle (EV) research is the production of sufficie (show more...)One of the greatest bottlenecks in extracellular vesicle (EV) research is the production of sufficient material in a consistent and effective way using in vitro cell models. Although the production of EVs in bioreactors maximizes EV yield in comparison to conventional cell cultures, the impact of their cell growth conditions on EVs has not yet been established. In this study, we grew two prostate cancer cell lines, PC-3 and VCaP, in conventional cell culture dishes and in two-chamber bioreactors to elucidate how the growth environment affects the EV characteristics. Specifically, we wanted to investigate the growth condition-dependent differences by non-targeted metabolite profiling using liquid chromatography–mass spectrometry (LC–MS) analysis. EVs were also characterized by their morphology, size distribution, and EV protein marker expression, and the EV yields were quantified by NTA. The use of bioreactor increased the EV yield >100 times compared to the conventional cell culture system. Regarding morphology, size distribution and surface markers, only minor differences were observed between the bioreactor-derived EVs (BR-EVs) and the EVs obtained from cells grown in conventional cell cultures (C-EVs). In contrast, metabolomic analysis revealed statistically significant differences in both polar and non-polar metabolites when the BR-EVs were compared to the C-EVs. The results show that the growth conditions markedly affected the EV metabolite profiles and that metabolomics was a sensitive tool to study molecular differences of EVs. We conclude that the cell culture conditions of EV production should be standardized and carefully detailed in publications and care should be taken when EVs from different production platforms are compared with each other for systemic effects. (hide) EV-METRIC 66% (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 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 Adj. k-factor 785.9 (pelleting) / 89.2 (washing) Protein markers EV: CD81/ TSG101/ CD29/ CD9 non-EV: calnexin Proteomics no Show all info Study aim Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells VCaP EV-harvesting Medium Serum-containing, but physical separation of serum EVs and secreted EVs (e.g. Bioreactor flask) 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 Yes Pelleting: time(min) 120 Pelleting: rotor type Type 50.2 Ti Pelleting: speed (g) 20000 Pelleting: adjusted k-factor 785.9 Wash: time (min) 120 Wash: Rotor Type TLA-55 Wash: speed (g) 100000 Wash: adjusted k-factor 89.20 Characterization: Protein analysis Protein Concentration Method Lowry-based assay Western Blot Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD9, CD81, TSG101, CD29 Not detected contaminants calnexin Characterization: Lipid analysis No Characterization: Particle analysis PMID previous EV particle analysis Electron microscopy Extra particle analysis NTA Report type Mean Reported size (nm) 122.7 EV concentration Yes EM EM-type Transmission-EM Image type Close-up

	EV180029	6/8	Homo sapiens	PC3	(d)(U)C	Palviainen, Mari	2019	66%
Study summary Full title Metabolic signature of extracellular vesicles depends on the cell culture conditions All authors Mari Palviainen ORCID Icon, Heikki Saari ORCID Icon, Olli Kärkkäinen ORCID Icon, Jenna Pekkinen, Seppo Auriola, Marjo Yliperttula, Maija Puhka, Kati Hanhineva & Pia R.-M. Siljander Journal J Extracell Vesicles Abstract One of the greatest bottlenecks in extracellular vesicle (EV) research is the production of sufficie (show more...)One of the greatest bottlenecks in extracellular vesicle (EV) research is the production of sufficient material in a consistent and effective way using in vitro cell models. Although the production of EVs in bioreactors maximizes EV yield in comparison to conventional cell cultures, the impact of their cell growth conditions on EVs has not yet been established. In this study, we grew two prostate cancer cell lines, PC-3 and VCaP, in conventional cell culture dishes and in two-chamber bioreactors to elucidate how the growth environment affects the EV characteristics. Specifically, we wanted to investigate the growth condition-dependent differences by non-targeted metabolite profiling using liquid chromatography–mass spectrometry (LC–MS) analysis. EVs were also characterized by their morphology, size distribution, and EV protein marker expression, and the EV yields were quantified by NTA. The use of bioreactor increased the EV yield >100 times compared to the conventional cell culture system. Regarding morphology, size distribution and surface markers, only minor differences were observed between the bioreactor-derived EVs (BR-EVs) and the EVs obtained from cells grown in conventional cell cultures (C-EVs). In contrast, metabolomic analysis revealed statistically significant differences in both polar and non-polar metabolites when the BR-EVs were compared to the C-EVs. The results show that the growth conditions markedly affected the EV metabolite profiles and that metabolomics was a sensitive tool to study molecular differences of EVs. We conclude that the cell culture conditions of EV production should be standardized and carefully detailed in publications and care should be taken when EVs from different production platforms are compared with each other for systemic effects. (hide) EV-METRIC 66% (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 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 Adj. k-factor 142.9 (pelleting) / 89.2 (washing) Protein markers EV: CD81/ TSG101/ CD29/ CD9 non-EV: calnexin Proteomics no Show all info Study aim Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells PC3 EV-harvesting Medium Serum-containing, but physical separation of serum EVs and secreted EVs (e.g. Bioreactor flask) 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) 120 Pelleting: rotor type Type 50.2 Ti Pelleting: speed (g) 110000 Pelleting: adjusted k-factor 142.9 Wash: time (min) 120 Wash: Rotor Type TLA-55 Wash: speed (g) 100000 Wash: adjusted k-factor 89.20 Characterization: Protein analysis Protein Concentration Method Lowry-based assay Western Blot Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD9, CD81, TSG101, CD29 Not detected contaminants calnexin Characterization: Lipid analysis No Characterization: Particle analysis PMID previous EV particle analysis Electron microscopy Extra particle analysis NTA Report type Mean Reported size (nm) 118.7 EV concentration Yes EM EM-type Transmission-EM Image type Wide-field

	EV180029	7/8	Homo sapiens	PC3	(d)(U)C	Palviainen, Mari	2019	66%
Study summary Full title Metabolic signature of extracellular vesicles depends on the cell culture conditions All authors Mari Palviainen ORCID Icon, Heikki Saari ORCID Icon, Olli Kärkkäinen ORCID Icon, Jenna Pekkinen, Seppo Auriola, Marjo Yliperttula, Maija Puhka, Kati Hanhineva & Pia R.-M. Siljander Journal J Extracell Vesicles Abstract One of the greatest bottlenecks in extracellular vesicle (EV) research is the production of sufficie (show more...)One of the greatest bottlenecks in extracellular vesicle (EV) research is the production of sufficient material in a consistent and effective way using in vitro cell models. Although the production of EVs in bioreactors maximizes EV yield in comparison to conventional cell cultures, the impact of their cell growth conditions on EVs has not yet been established. In this study, we grew two prostate cancer cell lines, PC-3 and VCaP, in conventional cell culture dishes and in two-chamber bioreactors to elucidate how the growth environment affects the EV characteristics. Specifically, we wanted to investigate the growth condition-dependent differences by non-targeted metabolite profiling using liquid chromatography–mass spectrometry (LC–MS) analysis. EVs were also characterized by their morphology, size distribution, and EV protein marker expression, and the EV yields were quantified by NTA. The use of bioreactor increased the EV yield >100 times compared to the conventional cell culture system. Regarding morphology, size distribution and surface markers, only minor differences were observed between the bioreactor-derived EVs (BR-EVs) and the EVs obtained from cells grown in conventional cell cultures (C-EVs). In contrast, metabolomic analysis revealed statistically significant differences in both polar and non-polar metabolites when the BR-EVs were compared to the C-EVs. The results show that the growth conditions markedly affected the EV metabolite profiles and that metabolomics was a sensitive tool to study molecular differences of EVs. We conclude that the cell culture conditions of EV production should be standardized and carefully detailed in publications and care should be taken when EVs from different production platforms are compared with each other for systemic effects. (hide) EV-METRIC 66% (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 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 Adj. k-factor 785.9 (pelleting) / 89.2 (washing) Protein markers EV: CD81/ TSG101/ CD29/ CD9 non-EV: calnexin Proteomics no Show all info Study aim Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells PC3 EV-harvesting Medium Serum-containing, but physical separation of serum EVs and secreted EVs (e.g. Bioreactor flask) 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 Yes Pelleting: time(min) 120 Pelleting: rotor type Type 50.2 Ti Pelleting: speed (g) 20000 Pelleting: adjusted k-factor 785.9 Wash: time (min) 120 Wash: Rotor Type TLA-55 Wash: speed (g) 100000 Wash: adjusted k-factor 89.20 Characterization: Protein analysis Protein Concentration Method Lowry-based assay Western Blot Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD9, CD81, TSG101, CD29 Not detected contaminants calnexin Characterization: Lipid analysis No Characterization: Particle analysis PMID previous EV particle analysis Electron microscopy Extra particle analysis NTA Report type Mean Reported size (nm) 148.8 EV concentration Yes EM EM-type Transmission-EM Image type Wide-field

	EV180029	8/8	Homo sapiens	VCaP	(d)(U)C	Palviainen, Mari	2019	66%
Study summary Full title Metabolic signature of extracellular vesicles depends on the cell culture conditions All authors Mari Palviainen ORCID Icon, Heikki Saari ORCID Icon, Olli Kärkkäinen ORCID Icon, Jenna Pekkinen, Seppo Auriola, Marjo Yliperttula, Maija Puhka, Kati Hanhineva & Pia R.-M. Siljander Journal J Extracell Vesicles Abstract One of the greatest bottlenecks in extracellular vesicle (EV) research is the production of sufficie (show more...)One of the greatest bottlenecks in extracellular vesicle (EV) research is the production of sufficient material in a consistent and effective way using in vitro cell models. Although the production of EVs in bioreactors maximizes EV yield in comparison to conventional cell cultures, the impact of their cell growth conditions on EVs has not yet been established. In this study, we grew two prostate cancer cell lines, PC-3 and VCaP, in conventional cell culture dishes and in two-chamber bioreactors to elucidate how the growth environment affects the EV characteristics. Specifically, we wanted to investigate the growth condition-dependent differences by non-targeted metabolite profiling using liquid chromatography–mass spectrometry (LC–MS) analysis. EVs were also characterized by their morphology, size distribution, and EV protein marker expression, and the EV yields were quantified by NTA. The use of bioreactor increased the EV yield >100 times compared to the conventional cell culture system. Regarding morphology, size distribution and surface markers, only minor differences were observed between the bioreactor-derived EVs (BR-EVs) and the EVs obtained from cells grown in conventional cell cultures (C-EVs). In contrast, metabolomic analysis revealed statistically significant differences in both polar and non-polar metabolites when the BR-EVs were compared to the C-EVs. The results show that the growth conditions markedly affected the EV metabolite profiles and that metabolomics was a sensitive tool to study molecular differences of EVs. We conclude that the cell culture conditions of EV production should be standardized and carefully detailed in publications and care should be taken when EVs from different production platforms are compared with each other for systemic effects. (hide) EV-METRIC 66% (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 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 Adj. k-factor 785.9 (pelleting) / 89.2 (washing) Protein markers EV: CD81/ TSG101/ CD29/ CD9 non-EV: calnexin Proteomics no Show all info Study aim Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells VCaP EV-harvesting Medium EV-depleted serum Preparation of EDS overnight (16h) at >=100,000g 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 Yes Pelleting: time(min) 120 Pelleting: rotor type Type 50.2 Ti Pelleting: speed (g) 20000 Pelleting: adjusted k-factor 785.9 Wash: time (min) 120 Wash: Rotor Type TLA-55 Wash: speed (g) 100000 Wash: adjusted k-factor 89.20 Characterization: Protein analysis Protein Concentration Method Lowry-based assay Western Blot Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD9, CD81, TSG101, CD29 Not detected contaminants calnexin Characterization: Lipid analysis No Characterization: Particle analysis PMID previous EV particle analysis Electron microscopy Extra particle analysis NTA Report type Mean Reported size (nm) 121.4 EV concentration Yes EM EM-type Transmission-EM Image type Wide-field

	EV180021	1/4	Homo sapiens	L540	(d)(U)C	Bachurski, Daniel	2019	66%
Study summary Full title Extracellular vesicle measurements with nanoparticle tracking analysis – An accuracy and repeatability comparison between NanoSight NS300 and ZetaView All authors Daniel Bachurski ORCID Icon, Maximiliane Schuldner, Phuong-Hien Nguyen, Alexandra Malz, Katrin S Reiners, Patricia C Grenzi ORCID Icon, Felix Babatz, Astrid C Schauss, Hinrich P Hansen, Michael Hallek & Elke Pogge von Strandmann Journal J Extracell Vesicles Abstract The expanding field of extracellular vesicle (EV) research needs reproducible and accurate methods t (show more...)The expanding field of extracellular vesicle (EV) research needs reproducible and accurate methods to characterize single EVs. Nanoparticle Tracking Analysis (NTA) is commonly used to determine EV concentration and diameter. As the EV field is lacking methods to easily confirm and validate NTA data, questioning the reliability of measurements remains highly important. In this regard, a comparison addressing measurement quality between different NTA devices such as Malvern’s NanoSight NS300 or Particle Metrix’ ZetaView has not yet been conducted. To evaluate the accuracy and repeatability of size and concentration determinations of both devices, we employed comparative methods including transmission electron microscopy (TEM) and single particle interferometric reflectance imaging sensing (SP-IRIS) by ExoView. Multiple test measurements with nanospheres, liposomes and ultracentrifuged EVs from human serum and cell culture supernatant were performed. Additionally, serial dilutions and freeze-thaw cycle-dependent EV decrease were measured to determine the robustness of each system. Strikingly, NanoSight NS300 exhibited a 2.0–2.1-fold overestimation of polystyrene and silica nanosphere concentration. By measuring serial dilutions of EV samples, we demonstrated higher accuracy in concentration determination by ZetaView (% BIAS range: 2.7–8.5) in comparison with NanoSight NS300 (% BIAS range: 32.9–36.8). The concentration measurements by ZetaView were also more precise (% CV range: 0.0–4.7) than measurements by NanoSight NS300 (% CV range: 5.4–10.7). On the contrary, quantitative TEM imaging indicated more accurate EV sizing by NanoSight NS300 (% DTEM range: 79.5–134.3) compared to ZetaView (% DTEM range: 111.8–205.7), while being equally repeatable (NanoSight NS300% CV range: 0.8–6.7; ZetaView: 1.4–7.8). However, both devices failed to report a peak EV diameter below 60 nm compared to TEM and SP-IRIS. Taken together, NTA devices differ strongly in their hardware and software affecting measuring results. ZetaView provided a more accurate and repeatable depiction of EV concentration, whereas NanoSight NS300 supplied size measurements of higher resolution. (hide) EV-METRIC 66% (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 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 Adj. k-factor 892 (washing) Protein markers EV: TSG101/ HSP70/ CD63/ CD9/ CD81 non-EV: Calnexin 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 L540 EV-harvesting Medium Serum free medium Cell viability (%) NA 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 Yes Pelleting: time(min) 30 Pelleting: speed (g) 10000 Wash: time (min) 30 Wash: Rotor Type TLA-55 Wash: speed (g) 10000 Wash: adjusted k-factor 892.0 Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD63, HSP70, TSG101 Not detected contaminants Calnexin Characterization: Lipid analysis Yes Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 100-500 EV concentration Yes EM EM-type Transmission-EM Image type Close-up, Wide-field Report size (nm) 30-200 Other particle analysis name(1) ExoView Report type Size range/distribution Report size 50-200 EV-concentration No Extra information EV-Track data set is associated with a technical paper comparing different NTA devices assessed by TEM and ExoView

	EV180021	2/4	Homo sapiens	Serum	(d)(U)C	Bachurski, Daniel	2019	66%
Study summary Full title Extracellular vesicle measurements with nanoparticle tracking analysis – An accuracy and repeatability comparison between NanoSight NS300 and ZetaView All authors Daniel Bachurski ORCID Icon, Maximiliane Schuldner, Phuong-Hien Nguyen, Alexandra Malz, Katrin S Reiners, Patricia C Grenzi ORCID Icon, Felix Babatz, Astrid C Schauss, Hinrich P Hansen, Michael Hallek & Elke Pogge von Strandmann Journal J Extracell Vesicles Abstract The expanding field of extracellular vesicle (EV) research needs reproducible and accurate methods t (show more...)The expanding field of extracellular vesicle (EV) research needs reproducible and accurate methods to characterize single EVs. Nanoparticle Tracking Analysis (NTA) is commonly used to determine EV concentration and diameter. As the EV field is lacking methods to easily confirm and validate NTA data, questioning the reliability of measurements remains highly important. In this regard, a comparison addressing measurement quality between different NTA devices such as Malvern’s NanoSight NS300 or Particle Metrix’ ZetaView has not yet been conducted. To evaluate the accuracy and repeatability of size and concentration determinations of both devices, we employed comparative methods including transmission electron microscopy (TEM) and single particle interferometric reflectance imaging sensing (SP-IRIS) by ExoView. Multiple test measurements with nanospheres, liposomes and ultracentrifuged EVs from human serum and cell culture supernatant were performed. Additionally, serial dilutions and freeze-thaw cycle-dependent EV decrease were measured to determine the robustness of each system. Strikingly, NanoSight NS300 exhibited a 2.0–2.1-fold overestimation of polystyrene and silica nanosphere concentration. By measuring serial dilutions of EV samples, we demonstrated higher accuracy in concentration determination by ZetaView (% BIAS range: 2.7–8.5) in comparison with NanoSight NS300 (% BIAS range: 32.9–36.8). The concentration measurements by ZetaView were also more precise (% CV range: 0.0–4.7) than measurements by NanoSight NS300 (% CV range: 5.4–10.7). On the contrary, quantitative TEM imaging indicated more accurate EV sizing by NanoSight NS300 (% DTEM range: 79.5–134.3) compared to ZetaView (% DTEM range: 111.8–205.7), while being equally repeatable (NanoSight NS300% CV range: 0.8–6.7; ZetaView: 1.4–7.8). However, both devices failed to report a peak EV diameter below 60 nm compared to TEM and SP-IRIS. Taken together, NTA devices differ strongly in their hardware and software affecting measuring results. ZetaView provided a more accurate and repeatable depiction of EV concentration, whereas NanoSight NS300 supplied size measurements of higher resolution. (hide) EV-METRIC 66% (95th 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 Serum 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 Adj. k-factor 892 (washing) Protein markers EV: HSP70/ CD63/ CD9 non-EV: Calnexin Proteomics no Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Serum 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 Yes Pelleting: time(min) 30 Pelleting: speed (g) 10000 Wash: time (min) 30 Wash: Rotor Type TLA-55 Wash: speed (g) 10000 Wash: adjusted k-factor 892.0 Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD63, HSP70 Not detected contaminants Calnexin Characterization: Lipid analysis Yes Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 100-500 EV concentration Yes EM EM-type Transmission-EM Image type Close-up, Wide-field Report size (nm) 30-500 Other particle analysis name(1) ExoView Report type Size range/distribution Report size 50-200 EV-concentration No Extra information EV-Track data set is associated with a technical paper comparing different NTA devices assessed by TEM and ExoView

	EV170032	1/2	Mus musculus	DC2.4, 4T1	(d)(U)C Flow cytometry	Aizea Morales-Kastresana	2019	66%
Study summary Full title High-fidelity detection and sorting of nanoscale vesicles in viral disease and cancer All authors Aizea Morales-Kastresana, Thomas A. Musich, Joshua A. Welsh, William Telford, Thorsten Demberg, James C. S. Wood, Marty Bigos, Carley D. Ross, Aliaksander Kachynski, Alan Dean, Edward J. Felton, Jonathan Van Dyke, John Tigges, Vasilis Toxavidis, David R. Parks, W. Roy Overton, Aparna H. Kesarwala, Gordon J. Freeman, Ariel Rosner, Stephen P. Perfetto, Lise Pasquet, Masaki Terabe, Katherine McKinnon, Veena Kapoor, Jane B. Trepel, Anu Puri, Hisataka Kobayashi, Bryant Yung, Xiaoyuan Chen, Peter Guion, Peter Choyke, Susan J. Knox, Ionita Ghiran, Marjorie Robert-Guroff, Jay A. Berzofsky and Jennifer C. Jones Journal J Extracell Vesicles Abstract Biological nanoparticles, including viruses and extracellular vesicles (EVs), are of interest to man (show more...)Biological nanoparticles, including viruses and extracellular vesicles (EVs), are of interest to many fields of medicine as biomarkers and mediators of or treatments for disease. However, exosomes and small viruses fall below the detection limits of conventional flow cytometers due to the overlap of particle-associated scattered light signals with the detection of background instrument noise from diffusely scattered light. To identify, sort, and study distinct subsets of EVs and other nanoparticles, as individual particles, we developed nanoscale Fluorescence Analysis and Cytometric Sorting (nanoFACS) methods to maximise information and material that can be obtained with high speed, high resolution flow cytometers. This nanoFACS method requires analysis of the instrument background noise (herein defined as the “reference noise”). With these methods, we demonstrate detection of tumour cell-derived EVs with specific tumour antigens using both fluorescence and scattered light parameters. We further validated the performance of nanoFACS by sorting two distinct HIV strains to >95% purity and confirmed the viability (infectivity) and molecular specificity (specific cell tropism) of biological nanomaterials sorted with nanoFACS. This nanoFACS method provides a unique way to analyse and sort functional EV- and viral-subsets with preservation of vesicular structure, surface protein specificity and RNA cargo activity. (hide) EV-METRIC 66% (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 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 Flow cytometry Adj. k-factor 156.9 (pelleting) / 41.45 (washing) Protein markers EV: Alix/ TSG101/ MHC2/ PSMA non-EV: None Proteomics no Show all info Study aim Function, New methodological development, Identification of content (omics approaches), Technical analysis comparing/optimizing EV-related methods Sample Species Mus musculus Sample Type Cell culture supernatant EV-producing cells DC2.4, 4T1 EV-harvesting Medium EV-depleted serum Preparation of EDS >=18h at >= 100,000g Cell viability (%) NA 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 Pelleting: adjusted k-factor 156.9 Wash: time (min) 120 Wash: Rotor Type TLA-120.1 Wash: speed (g) 100000 Wash: adjusted k-factor 41.45 Fluorescence-activated vesicle sorting Type of flow cytometer Astrios-EQ, using combinations of protein, membrane, and epitope-specific labels Hardware adaptation to ~100nm EV's Yes Size of calibration beads (µm) 0.1, 0.2 Fluorescent labeling Specific labelling of EV conte Other Name other separation method Flow cytometry EV-subtype Used subtypes Yes Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins Alix, TSG101 Flow cytometry specific beads Antibody details provided? No Antibody dilution provided? No Selected surface protein(s) PSMA Flow cytometry Type of Flow cytometry Astrios-EQ Hardware adaptation to ~100nm EV's configuration with appropriate scatter thresholds and other settings for nanoFACS (high sensitivity optical path and signal processing) Calibration bead size 0.1, 0.2, 0.5 Antibody details provided? No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 80-180 EV concentration Yes Particle yield 5.00E+11 particles/ml start sample Particle analysis: flow cytometry Flow cytometer type Astrios-EQ Hardware adjustment configuration with appropriate scatter thresholds and other settings for nanoFACS (high sensitivity optical path and signal processing) Calibration bead size 0.1;0.2;0.5 and various Report type Median EV concentration Yes EM EM-type Transmission-EM Image type Close-up, Wide-field Extra information EVs purified with the serial ultracentrifugation methods were labeled with small molecules or antibodies, and excess/unbound small molecules and antibodies were removed with either NAP-5 or qEV size exclusion chromatography

	EV170032	2/2	Homo sapiens	PC3	(d)(U)C Flow cytometry	Aizea Morales-Kastresana	2019	66%
Study summary Full title High-fidelity detection and sorting of nanoscale vesicles in viral disease and cancer All authors Aizea Morales-Kastresana, Thomas A. Musich, Joshua A. Welsh, William Telford, Thorsten Demberg, James C. S. Wood, Marty Bigos, Carley D. Ross, Aliaksander Kachynski, Alan Dean, Edward J. Felton, Jonathan Van Dyke, John Tigges, Vasilis Toxavidis, David R. Parks, W. Roy Overton, Aparna H. Kesarwala, Gordon J. Freeman, Ariel Rosner, Stephen P. Perfetto, Lise Pasquet, Masaki Terabe, Katherine McKinnon, Veena Kapoor, Jane B. Trepel, Anu Puri, Hisataka Kobayashi, Bryant Yung, Xiaoyuan Chen, Peter Guion, Peter Choyke, Susan J. Knox, Ionita Ghiran, Marjorie Robert-Guroff, Jay A. Berzofsky and Jennifer C. Jones Journal J Extracell Vesicles Abstract Biological nanoparticles, including viruses and extracellular vesicles (EVs), are of interest to man (show more...)Biological nanoparticles, including viruses and extracellular vesicles (EVs), are of interest to many fields of medicine as biomarkers and mediators of or treatments for disease. However, exosomes and small viruses fall below the detection limits of conventional flow cytometers due to the overlap of particle-associated scattered light signals with the detection of background instrument noise from diffusely scattered light. To identify, sort, and study distinct subsets of EVs and other nanoparticles, as individual particles, we developed nanoscale Fluorescence Analysis and Cytometric Sorting (nanoFACS) methods to maximise information and material that can be obtained with high speed, high resolution flow cytometers. This nanoFACS method requires analysis of the instrument background noise (herein defined as the “reference noise”). With these methods, we demonstrate detection of tumour cell-derived EVs with specific tumour antigens using both fluorescence and scattered light parameters. We further validated the performance of nanoFACS by sorting two distinct HIV strains to >95% purity and confirmed the viability (infectivity) and molecular specificity (specific cell tropism) of biological nanomaterials sorted with nanoFACS. This nanoFACS method provides a unique way to analyse and sort functional EV- and viral-subsets with preservation of vesicular structure, surface protein specificity and RNA cargo activity. (hide) EV-METRIC 66% (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 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 Flow cytometry Adj. k-factor 156.9 (pelleting) / 41.45 (washing) Protein markers EV: Alix/ TSG101/ MHC2/ PSMA non-EV: None Proteomics no Show all info Study aim Function, New methodological development, Identification of content (omics approaches), Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells PC3 EV-harvesting Medium EV-depleted serum Preparation of EDS >=18h at >= 100,000g Cell viability (%) NA 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 Pelleting: adjusted k-factor 156.9 Wash: time (min) 120 Wash: Rotor Type TLA-120.1 Wash: speed (g) 100000 Wash: adjusted k-factor 41.45 Fluorescence-activated vesicle sorting Type of flow cytometer Astrios-EQ, using combinations of protein, membrane, and epitope-specific labels Hardware adaptation to ~100nm EV's Yes Size of calibration beads (µm) 0.1, 0.2 Fluorescent labeling Specific labelling of EV conte Other Name other separation method Flow cytometry EV-subtype Used subtypes Yes Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins Alix, TSG101 Flow cytometry specific beads Antibody details provided? No Antibody dilution provided? No Selected surface protein(s) PSMA Flow cytometry Type of Flow cytometry Astrios-EQ Hardware adaptation to ~100nm EV's configuration with appropriate scatter thresholds and other settings for nanoFACS (high sensitivity optical path and signal processing) Calibration bead size 0.1, 0.2, 0.5 Antibody details provided? No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 80-180 EV concentration Yes Particle yield 5.00E+11 particles/ml start sample Particle analysis: flow cytometry Flow cytometer type Astrios-EQ Hardware adjustment configuration with appropriate scatter thresholds and other settings for nanoFACS (high sensitivity optical path and signal processing) Calibration bead size 0.1;0.2;0.5 and various Report type Median EV concentration Yes EM EM-type Transmission-EM Image type Close-up, Wide-field Extra information EVs purified with the serial ultracentrifugation methods were labeled with small molecules or antibodies, and excess/unbound small molecules and antibodies were removed with either NAP-5 or qEV size exclusion chromatography

	EV190021	1/4	Homo sapiens	Blood plasma	(d)(U)C UF	Yunusova, Natalia	2019	63%
Study summary Full title Metalloproteinases at the surface of small extrcellular vesicles in advanced ovarian cancer: Relationships with ascites volume and peritoneal canceromatosis index All authors Natalia V Yunusova, Marina R Patysheva, Sergey V Molchanov, Elena A Zambalova, Alina E Grigor'eva, Larisa A Kolomiets, Maxim O Ochirov, Svetlana N Tamkovich, Irina V Kondakova Journal Clinica Chimica Acta Abstract Metalloproteinases and their extracellular matrix metalloproteinase inducer (EMMPRIN) play an essent (show more...)Metalloproteinases and their extracellular matrix metalloproteinase inducer (EMMPRIN) play an essential role in the regulation of signaling from growth factors receptors and adhesion molecules, cell motility and extracellular matrix degradation. The aim of the study was to evaluate the relationship between the levels of small extracellular vesicles (sEVs) metalloproteinases, such as ADAM10, ADAM17, MMP2, MMP9 and EMMPRIN and ascites volume and peritoneal canceromatosis index in advanced ovarian cancer patients (OCPs). The subpopulations of metalloproteinases at the surface of sEVs of borderline ovarian tumor patients (BOTPs) (n = 20, 36.5 ± 2.5 years) and previously untreated advanced OCPs (n = 35, 56.5 ± 2.5 years) were evaluated using flow cytometry. The metalloproteinase subpopulations of CD9-positive sEVs isolated from plasma of BOTPs and OCPs appeared to be quite similar. However, a significant difference in the expression of ADAM-metalloproteinases in ascites sEVs was found between BOTPs and OCPs. The level of sEVs metalloproteinases in OCPs significantly depended on the ascites volume. A statistically significant relationship between the level of ADAM10+/ADAM17- subpopulation in plasma sEVs and the peritoneal canceromatosis index was found (R = 0.66, p < .05). The levels of metalloproteinases and EMMPRIN in circulating sEVs, as well as the assessment of individual subpopulations may be promising approaches to OCPs managing. (hide) EV-METRIC 63% (91st 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 borderline ovarian tumors, control subjects 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 UF Protein markers EV: CD81/ CD63/ CD9/ CD24 non-EV: / Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Blood plasma 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) 90 Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 16 Wash: time (min) 90 Wash: Rotor Type SW 32 Ti Wash: speed (g) 1000000 Ultra filtration Cut-off size (kDa) 100 Membrane type Polyethersulfone (PES) Characterization: Protein analysis Protein Concentration Method Fluorometric assay (e.g. Qubit, NanoOrange,...) Flow cytometry specific beads Antibody details provided? No Antibody dilution provided? No Detected EV-associated proteins CD24/ CD9/ CD63/ CD81 Not detected EV-associated proteins CD81/ CD63/ CD9/ CD24 Detected contaminants Not detected contaminants Characterization: Lipid analysis No EM EM-type Transmission-EM Image type Close-up, Wide-field Report size (nm) 100

	EV190004	1/1	Homo sapiens	Urine	qEV UF	Eline Oeyen	2019	63%
Study summary Full title Determination of variability due to biological and technical variation in urinary extracellular vesicles as a crucial step in biomarker discovery studies All authors Eline Oeyen, Hanny Willems, Ruben T Kindt, Koen Sandra, Kurt Boonen, Lucien Hoekx, Stefan De Wachter, Filip Ameye and Inge Mertensa Journal J Extracell Vesicles Abstract Urinary extracellular vesicles (EVs) are an attractive source of biomarkers for urological diseases. (show more...)Urinary extracellular vesicles (EVs) are an attractive source of biomarkers for urological diseases. A crucial step in biomarker discovery studies is the determination of the variation parameters to perform a sample size calculation. In this way, a biomarker discovery study with sufficient statistical power can be performed to obtain biologically significant biomarkers. Here, a variation study was performed on both the protein and lipid content of urinary EVs of healthy individuals, aged between 52 and 69 years. Ultrafiltration (UF) in combination with size exclusion chromatography (SEC) was used to isolate the EVs from urine. Different experimental variation set-ups were used in this variation study. The calculated standard deviations (SDs) of the 90% least variable peptides and lipids did not exceed 2 and 1.2, respectively. These parameters can be used in a sample size calculation for a well-designed biomarker discovery study at the cargo of EVs. (hide) EV-METRIC 63% (92nd 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 Urine 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 qEV UF Protein markers EV: Flotillin1 non-EV: Proteomics yes Show all info Study aim Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Urine Separation Method Ultra filtration Cut-off size (kDa) 100 Membrane type Regenerated cellulose Commercial kit qEV Other Name other separation method qEV Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? No Detected EV-associated proteins Flotillin1 Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 108 EV concentration Yes Particle analysis: flow cytometry Hardware adjustment EM EM-type Transmission-EM Image type Close-up, Wide-field Report size (nm) 40-100 Report type Size range/distribution Report size 140-160 EV-concentration No

	EV180071	1/3	Homo sapiens	Blood plasma	SEC UF	Brahmer A	2019	63%
Study summary Full title Platelets, endothelial cells and leukocytes contribute to the exercise-triggered release of extracellular vesicles into the circulation. All authors Brahmer A, Neuberger E, Esch-Heisser L, Haller N, Jorgensen MM, Baek R, Möbius W, Simon P, Krämer-Albers EM. Journal J Extracell Vesicles Abstract Physical activity initiates a wide range of multi-systemic adaptations that promote mental and physi (show more...)Physical activity initiates a wide range of multi-systemic adaptations that promote mental and physical health. Recent work demonstrated that exercise triggers the release of extracellular vesicles (EVs) into the circulation, possibly contributing to exercise-associated adaptive systemic signalling. Circulating EVs comprise a heterogeneous collection of different EV-subclasses released from various cell types. So far, a comprehensive picture of the parental and target cell types, EV-subpopulation diversity and functional properties of EVs released during exercise (ExerVs) is lacking. Here, we performed a detailed EV-phenotyping analysis to explore the cellular origin and potential subtypes of ExerVs. Healthy male athletes were subjected to an incremental cycling test until exhaustion and blood was drawn before, during, and immediately after the test. Analysis of total blood plasma by EV Array suggested endothelial and leukocyte characteristics of ExerVs. We further purified ExerVs from plasma by size exclusion chromatography as well as CD9-, CD63- or CD81-immunobead isolation to examine ExerV-subclass dynamics. EV-marker analysis demonstrated increasing EV-levels during cycling exercise, with highest levels at peak exercise in all EV-subclasses analysed. Phenotyping of ExerVs using a multiplexed flow-cytometry platform revealed a pattern of cell surface markers associated with ExerVs and identified lymphocytes (CD4, CD8), monocytes (CD14), platelets (CD41, CD42, CD62P), endothelial cells (CD105, CD146) and antigen presenting cells (MHC-II) as ExerV-parental cells. We conclude that multiple cell types associated with the circulatory system contribute to a pool of heterogeneous ExerVs, which may be involved in exercise-related signalling mechanisms and tissue crosstalk. (hide) EV-METRIC 63% (91st 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 SEC UF Protein markers EV: TSG101/ CD31/ CD209/ CD326/ CD133/1/ CD8/ CD9/ CD49e/ CD81/ CD86/ Syntenin/ CD41b/ CD29/ CD63/ CD42a/ CD44/ CD20/ CD40/ Sarcoglycan-alpha/ CD24/ CD146/ CD69/ MHC2/ ROR1/ MHC1/ SSEA4/ CD105/ MCSP/ CD62p/ CD19/ CD142 non-EV: / ApoA1 Proteomics no Show all info Study aim Biogenesis/cargo sorting/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Blood plasma Separation Method Ultra filtration Cut-off size (kDa) 30 Membrane type Regenerated cellulose Commercial kit Size-exclusion chromatography Total column volume (mL) 10 Sample volume/column (mL) 2 Resin type Sepharose CL-2B Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? No Detected EV-associated proteins CD81/ CD9/ TSG101/ Syntenin/ CD41b/ CD63 Not detected EV-associated proteins Sarcoglycan-alpha Detected contaminants ApoA1 Not detected contaminants Detected EV-associated proteins CD63/ CD9/ CD81/ CD8/ CD19/ CD20/ CD24/ CD29/ CD31/ CD40/ CD41b/ CD42a/ CD44/ CD49e/ CD62p/ CD69/ CD86/ CD105/ CD133/1/ CD142/ CD146/ CD209/ CD326/ MHC1/ MHC2/ MCSP/ ROR1/ SSEA4 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 106 EM EM-type Transmission-EM Image type Wide-field

	EV180050	3/6	Homo sapiens	bone marrow-derived mesenchymal stem cells	(d)(U)C Filtration SEC UF	Alice Gualerzi	2019	62%
Study summary Full title Raman spectroscopy as a quick tool to assess purity of extracellular vesicle preparations and predict their functionality All authors Alice Gualerzi, Sander Alexander Antonius Kooijmans, Stefania Niada, Silvia Picciolini, Anna Teresa Brini, Giovanni Camussi & Marzia Bedoni Journal J Extracell Vesicles Abstract Extracellular vesicles (EVs) from a variety of stem cell sources are believed to harbour regenerativ (show more...)Extracellular vesicles (EVs) from a variety of stem cell sources are believed to harbour regenerative capacity, which may be exploited for therapeutic purposes. Because of EV interaction with other soluble secreted factors, EV activity may depend on the employed purification method, which limits cross-study comparisons and therapeutic development. Raman spectroscopy (RS) is a quick and easy method to assess EV purity and composition, giving in-depth biochemical overview on EV preparation. Hereby, we show how this method can be used to characterise EVs isolated from human liver stem cells and bone marrow mesenchymal stem/stromal cells by means of conventional ultracentrifugation (UC) and size exclusion chromatography (SEC) protocols. The obtained EV preparations were demonstrated to be characterised by different degrees of purity and a specific Raman fingerprint that represents both the cell source and the isolation procedure used. Moreover, RS provided useful hints to explore the factors underlying the functional diversity of EV preparations from the same cell source, thus representing a valuable tool to assess EV quality prior to functional assays or therapeutic application. (hide) EV-METRIC 62% (92nd 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 SEC UF Protein markers EV: CD81/ Flotillin-1/ CD63 non-EV: Calnexin/ Calreticulin Proteomics no Show all info Study aim New methodological development, Technical analysis comparing/optimizing EV-related methods, Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells bone marrow-derived mesenchymal stem cells 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 Pelleting performed No Filtration steps > 0.45 µm, Ultra filtration Cut-off size (kDa) 100 Membrane type Not specified Size-exclusion chromatography Total column volume (mL) 120 Sample volume/column (mL) 2 Resin type HiPrep 16/60 Sephacryl S-400 HR Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD63, CD81, Flotillin-1 Not detected contaminants Calnexin, Calreticulin Characterization: Lipid analysis No Characterization: Particle analysis PMID previous EV particle analysis Other Extra particle analysis NTA Report type Mean Reported size (nm) 247 ± 68 EV concentration Yes EM EM-type Transmission-EM Image type Wide-field Other particle analysis name(1) Raman spectroscopy

	EV180050	4/6	Homo sapiens	liver stem cells	(d)(U)C Filtration SEC UF	Alice Gualerzi	2019	62%
Study summary Full title Raman spectroscopy as a quick tool to assess purity of extracellular vesicle preparations and predict their functionality All authors Alice Gualerzi, Sander Alexander Antonius Kooijmans, Stefania Niada, Silvia Picciolini, Anna Teresa Brini, Giovanni Camussi & Marzia Bedoni Journal J Extracell Vesicles Abstract Extracellular vesicles (EVs) from a variety of stem cell sources are believed to harbour regenerativ (show more...)Extracellular vesicles (EVs) from a variety of stem cell sources are believed to harbour regenerative capacity, which may be exploited for therapeutic purposes. Because of EV interaction with other soluble secreted factors, EV activity may depend on the employed purification method, which limits cross-study comparisons and therapeutic development. Raman spectroscopy (RS) is a quick and easy method to assess EV purity and composition, giving in-depth biochemical overview on EV preparation. Hereby, we show how this method can be used to characterise EVs isolated from human liver stem cells and bone marrow mesenchymal stem/stromal cells by means of conventional ultracentrifugation (UC) and size exclusion chromatography (SEC) protocols. The obtained EV preparations were demonstrated to be characterised by different degrees of purity and a specific Raman fingerprint that represents both the cell source and the isolation procedure used. Moreover, RS provided useful hints to explore the factors underlying the functional diversity of EV preparations from the same cell source, thus representing a valuable tool to assess EV quality prior to functional assays or therapeutic application. (hide) EV-METRIC 62% (92nd 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 SEC UF Protein markers EV: TSG101/ CD63/ CD81/ Alix/ beta-actin/ Flotillin-1/ CD9 non-EV: Calnexin/ Calreticulin Proteomics no Show all info Study aim New methodological development, Technical analysis comparing/optimizing EV-related methods, Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells liver stem cells 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 Pelleting performed No Filtration steps > 0.45 µm, Ultra filtration Cut-off size (kDa) 100 Membrane type Not specified Size-exclusion chromatography Total column volume (mL) 120 Sample volume/column (mL) 2 Resin type HiPrep 16/60 Sephacryl S-400 HR Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins Alix, CD63, CD81, CD9, Flotillin-1, TSG101, beta-actin Not detected contaminants Calnexin, Calreticulin Characterization: Lipid analysis No Characterization: Particle analysis PMID previous EV particle analysis Other Extra particle analysis NTA Report type Mean Reported size (nm) 228 ± 50 EV concentration Yes EM EM-type Transmission-EM Image type Wide-field Other particle analysis name(1) Raman spectroscopy

	EV170040	1/2	Rattus norvegicus	MTPa, MTPa-Tspan8	(d)(U)C Filtration UF	Voglstaetter, Maren	2019	62%
Study summary Full title Tspan8 is expressed in breast cancer and regulates E‐cadherin/catenin signalling and metastasis accompanied by increased circulating extracellular vesicles All authors Maren Voglstaetter, Andreas R Thomsen, Jerome Nouvel, Arend Koch, Paul Jank, Elena Grueso Navarro, Tanja Gainey‐Schleicher, Richa Khanduri, Andrea Groß, Florian Rossner, Carina Blaue, Clemens M Franz, Marina Veil, Gerhard Puetz, Andreas Hippe, Jochen Dindorf, Jubin Kashef, Wilko Thiele, Bernhard Homey, Celine Greco, Claude Boucheix, Andreas Baur, Thalia Erbes, Cornelius F Waller, Marie Follo, Ghamartaj Hossein, Christine Sers, Jonathan Sleeman, Irina Nazarenko Journal J Pathol Abstract Tspan8 exhibits a functional role in many cancer types including pancreatic, colorectal, oesophagus (show more...)Tspan8 exhibits a functional role in many cancer types including pancreatic, colorectal, oesophagus carcinoma, and melanoma. We present a first study on the expression and function of Tspan8 in breast cancer. Tspan8 protein was present in the majority of human primary breast cancer lesions and metastases in the brain, bone, lung, and liver. In a syngeneic rat breast cancer model, Tspan8+ tumours formed multiple liver and spleen metastases, while Tspan8− tumours exhibited a significantly diminished ability to metastasise, indicating a role of Tspan8 in metastases. Addressing the underlying molecular mechanisms, we discovered that Tspan8 can mediate up‐regulation of E‐cadherin and down‐regulation of Twist, p120‐catenin, and β‐catenin target genes accompanied by the change of cell phenotype, resembling the mesenchymal–epithelial transition. Furthermore, Tspan8+ cells exhibited enhanced cell–cell adhesion, diminished motility, and decreased sensitivity to irradiation. As a regulator of the content and function of extracellular vesicles (EVs), Tspan8 mediated a several‐fold increase in EV number in cell culture and the circulation of tumour‐bearing animals. We observed increased protein levels of E‐cadherin and p120‐catenin in these EVs; furthermore, Tspan8 and p120‐catenin were co‐immunoprecipitated, indicating that they may interact with each other. Altogether, our findings show the presence of Tspan8 in breast cancer primary lesion and metastases and indicate its role as a regulator of cell behaviour and EV release in breast cancer. © 2019 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland. (hide) EV-METRIC 62% (92nd 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 UF Protein markers EV: TSG101/ E-Cadherin/ Tspan8/ CD63/ CD9 non-EV: None Proteomics no Show all info Study aim Function Sample Species Rattus norvegicus Sample Type Cell culture supernatant EV-producing cells MTPa, MTPa-Tspan8 EV-harvesting Medium Serum free medium Cell viability (%) NA 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 No Filtration steps 0.22µm or 0.2µm Ultra filtration Cut-off size (kDa) 300 Membrane type Polyethersulfone (PES) Characterization: Protein analysis Protein Concentration Method microBCA Protein Yield (µg) 0.1 Western Blot Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD9, TSG101 Other 1 Co-immunoprecipitation Characterization: Lipid analysis No Characterization: Particle analysis DLS Report type particle size distribution by intensity and mass are reported Reported size (nm) 100 NTA Report type Size range/distribution Reported size (nm) 120 EV concentration Yes Particle yield 6.00E+10 particles/million cells EM EM-type Transmission-EM Image type Close-up, Wide-field

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