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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV210139	1/6	Homo sapiens	U87	(d)(U)C Filtration	Haraszti, Reka A	2016	44%
Study summary Full title High-resolution proteomic and lipidomic analysis of exosomes and microvesicles from different cell sources All authors Reka A Haraszti, Marie-Cecile Didiot, Ellen Sapp, John Leszyk, Scott A Shaffer, Hannah E Rockwell, Fei Gao, Niven R Narain, Marian DiFiglia, Michael A Kiebish, Neil Aronin, Anastasia Khvorova Journal J Extracell Vesicles Abstract Extracellular vesicles (EVs), including exosomes and microvesicles (MVs), are explored for use in di (show more...)Extracellular vesicles (EVs), including exosomes and microvesicles (MVs), are explored for use in diagnostics, therapeutics and drug delivery. However, little is known about the relationship of protein and lipid composition of EVs and their source cells. Here, we report high-resolution lipidomic and proteomic analyses of exosomes and MVs derived by differential ultracentrifugation from 3 different cell types: U87 glioblastoma cells, Huh7 hepatocellular carcinoma cells and human bone marrow-derived mesenchymal stem cells (MSCs). We identified 3,532 proteins and 1,961 lipid species in the screen. Exosomes differed from MVs in several different areas: (a) The protein patterns of exosomes were more likely different from their cells of origin than were the protein patterns of MVs; (b) The proteomes of U87 and Huh7 exosomes were similar to each other but different from the proteomes of MSC exosomes, whereas the lipidomes of Huh7 and MSC exosomes were similar to each other but different from the lipidomes of U87 exosomes; (c) exosomes exhibited proteins of extracellular matrix, heparin-binding, receptors, immune response and cell adhesion functions, whereas MVs were enriched in endoplasmic reticulum, proteasome and mitochondrial proteins. Exosomes and MVs also differed in their types of lipid contents. Enrichment in glycolipids and free fatty acids characterized exosomes, whereas enrichment in ceramides and sphingomyelins characterized MVs. Furthermore, Huh7 and MSC exosomes were specifically enriched in cardiolipins; U87 exosomes were enriched in sphingomyelins. This study comprehensively analyses the protein and lipid composition of exosomes, MVs and source cells in 3 different cell types. (hide) EV-METRIC 44% (84th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles exosome Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Filtration Protein markers EV: CD81/ TSG101/ CD63/ CD9 non-EV: Calnexin Proteomics yes Show all info Study aim Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells U87 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 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 Not specified Pelleting: speed (g) 10000 Filtration steps 0.22µm or 0.2µmNo Characterization: Protein analysis Protein Concentration Method Bradford Western Blot Detected EV-associated proteins CD9/ CD63/ CD81 Not detected EV-associated proteins Tsg101 Not detected contaminants Calnexin Proteomics database No Characterization: Lipid analysis Yes Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 0-500 EV concentration Yes Particle yield Not reported NA EM EM-type Transmission-EM Image type Wide-field

	EV210139	2/6	Homo sapiens	U87	(d)(U)C	Haraszti, Reka A	2016	44%
Study summary Full title High-resolution proteomic and lipidomic analysis of exosomes and microvesicles from different cell sources All authors Reka A Haraszti, Marie-Cecile Didiot, Ellen Sapp, John Leszyk, Scott A Shaffer, Hannah E Rockwell, Fei Gao, Niven R Narain, Marian DiFiglia, Michael A Kiebish, Neil Aronin, Anastasia Khvorova Journal J Extracell Vesicles Abstract Extracellular vesicles (EVs), including exosomes and microvesicles (MVs), are explored for use in di (show more...)Extracellular vesicles (EVs), including exosomes and microvesicles (MVs), are explored for use in diagnostics, therapeutics and drug delivery. However, little is known about the relationship of protein and lipid composition of EVs and their source cells. Here, we report high-resolution lipidomic and proteomic analyses of exosomes and MVs derived by differential ultracentrifugation from 3 different cell types: U87 glioblastoma cells, Huh7 hepatocellular carcinoma cells and human bone marrow-derived mesenchymal stem cells (MSCs). We identified 3,532 proteins and 1,961 lipid species in the screen. Exosomes differed from MVs in several different areas: (a) The protein patterns of exosomes were more likely different from their cells of origin than were the protein patterns of MVs; (b) The proteomes of U87 and Huh7 exosomes were similar to each other but different from the proteomes of MSC exosomes, whereas the lipidomes of Huh7 and MSC exosomes were similar to each other but different from the lipidomes of U87 exosomes; (c) exosomes exhibited proteins of extracellular matrix, heparin-binding, receptors, immune response and cell adhesion functions, whereas MVs were enriched in endoplasmic reticulum, proteasome and mitochondrial proteins. Exosomes and MVs also differed in their types of lipid contents. Enrichment in glycolipids and free fatty acids characterized exosomes, whereas enrichment in ceramides and sphingomyelins characterized MVs. Furthermore, Huh7 and MSC exosomes were specifically enriched in cardiolipins; U87 exosomes were enriched in sphingomyelins. This study comprehensively analyses the protein and lipid composition of exosomes, MVs and source cells in 3 different cell types. (hide) EV-METRIC 44% (84th 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 (shedding) microvesicle 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/ TSG101/ CD63/ CD9 non-EV: Calnexin Proteomics yes Show all info Study aim Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells U87 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 10,000 g and 50,000 g Pelleting performed Yes Pelleting: time(min) 30 Pelleting: rotor type Not specified Pelleting: speed (g) 10000 Characterization: Protein analysis Protein Concentration Method Bradford Western Blot Detected EV-associated proteins CD9/ CD63/ CD81 Not detected EV-associated proteins Tsg101 Detected contaminants Calnexin Proteomics database No Characterization: Lipid analysis Yes Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 0-600 EV concentration Yes Particle yield Not reported NA EM EM-type Transmission-EM Image type Wide-field

	EV210139	3/6	Homo sapiens	Huh7	(d)(U)C Filtration	Haraszti, Reka A	2016	44%
Study summary Full title High-resolution proteomic and lipidomic analysis of exosomes and microvesicles from different cell sources All authors Reka A Haraszti, Marie-Cecile Didiot, Ellen Sapp, John Leszyk, Scott A Shaffer, Hannah E Rockwell, Fei Gao, Niven R Narain, Marian DiFiglia, Michael A Kiebish, Neil Aronin, Anastasia Khvorova Journal J Extracell Vesicles Abstract Extracellular vesicles (EVs), including exosomes and microvesicles (MVs), are explored for use in di (show more...)Extracellular vesicles (EVs), including exosomes and microvesicles (MVs), are explored for use in diagnostics, therapeutics and drug delivery. However, little is known about the relationship of protein and lipid composition of EVs and their source cells. Here, we report high-resolution lipidomic and proteomic analyses of exosomes and MVs derived by differential ultracentrifugation from 3 different cell types: U87 glioblastoma cells, Huh7 hepatocellular carcinoma cells and human bone marrow-derived mesenchymal stem cells (MSCs). We identified 3,532 proteins and 1,961 lipid species in the screen. Exosomes differed from MVs in several different areas: (a) The protein patterns of exosomes were more likely different from their cells of origin than were the protein patterns of MVs; (b) The proteomes of U87 and Huh7 exosomes were similar to each other but different from the proteomes of MSC exosomes, whereas the lipidomes of Huh7 and MSC exosomes were similar to each other but different from the lipidomes of U87 exosomes; (c) exosomes exhibited proteins of extracellular matrix, heparin-binding, receptors, immune response and cell adhesion functions, whereas MVs were enriched in endoplasmic reticulum, proteasome and mitochondrial proteins. Exosomes and MVs also differed in their types of lipid contents. Enrichment in glycolipids and free fatty acids characterized exosomes, whereas enrichment in ceramides and sphingomyelins characterized MVs. Furthermore, Huh7 and MSC exosomes were specifically enriched in cardiolipins; U87 exosomes were enriched in sphingomyelins. This study comprehensively analyses the protein and lipid composition of exosomes, MVs and source cells in 3 different cell types. (hide) EV-METRIC 44% (84th 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 (shedding) microvesicle 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: CD81/ TSG101/ CD63/ CD9 non-EV: Calnexin Proteomics yes Show all info Study aim Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Huh7 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 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 Not specified Pelleting: speed (g) 10000 Filtration steps 0.22µm or 0.2µmNo Characterization: Protein analysis Protein Concentration Method Bradford Western Blot Detected EV-associated proteins CD9/ CD63/ CD81/ Tsg101 Not detected contaminants Calnexin Proteomics database No Characterization: Lipid analysis Yes Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 0-500 EV concentration Yes Particle yield Not reported NA EM EM-type Transmission-EM Image type Wide-field

	EV210139	4/6	Homo sapiens	Huh7	(d)(U)C	Haraszti, Reka A	2016	44%
Study summary Full title High-resolution proteomic and lipidomic analysis of exosomes and microvesicles from different cell sources All authors Reka A Haraszti, Marie-Cecile Didiot, Ellen Sapp, John Leszyk, Scott A Shaffer, Hannah E Rockwell, Fei Gao, Niven R Narain, Marian DiFiglia, Michael A Kiebish, Neil Aronin, Anastasia Khvorova Journal J Extracell Vesicles Abstract Extracellular vesicles (EVs), including exosomes and microvesicles (MVs), are explored for use in di (show more...)Extracellular vesicles (EVs), including exosomes and microvesicles (MVs), are explored for use in diagnostics, therapeutics and drug delivery. However, little is known about the relationship of protein and lipid composition of EVs and their source cells. Here, we report high-resolution lipidomic and proteomic analyses of exosomes and MVs derived by differential ultracentrifugation from 3 different cell types: U87 glioblastoma cells, Huh7 hepatocellular carcinoma cells and human bone marrow-derived mesenchymal stem cells (MSCs). We identified 3,532 proteins and 1,961 lipid species in the screen. Exosomes differed from MVs in several different areas: (a) The protein patterns of exosomes were more likely different from their cells of origin than were the protein patterns of MVs; (b) The proteomes of U87 and Huh7 exosomes were similar to each other but different from the proteomes of MSC exosomes, whereas the lipidomes of Huh7 and MSC exosomes were similar to each other but different from the lipidomes of U87 exosomes; (c) exosomes exhibited proteins of extracellular matrix, heparin-binding, receptors, immune response and cell adhesion functions, whereas MVs were enriched in endoplasmic reticulum, proteasome and mitochondrial proteins. Exosomes and MVs also differed in their types of lipid contents. Enrichment in glycolipids and free fatty acids characterized exosomes, whereas enrichment in ceramides and sphingomyelins characterized MVs. Furthermore, Huh7 and MSC exosomes were specifically enriched in cardiolipins; U87 exosomes were enriched in sphingomyelins. This study comprehensively analyses the protein and lipid composition of exosomes, MVs and source cells in 3 different cell types. (hide) EV-METRIC 44% (84th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles exosome Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Protein markers EV: CD81/ TSG101/ CD63/ CD9 non-EV: Calnexin Proteomics yes Show all info Study aim Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Huh7 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 10,000 g and 50,000 g Pelleting performed Yes Pelleting: time(min) 30 Pelleting: rotor type Not specified Pelleting: speed (g) 10000 Characterization: Protein analysis Protein Concentration Method Bradford Western Blot Detected EV-associated proteins CD9/ CD63/ CD81 Not detected EV-associated proteins Tsg101 Detected contaminants Calnexin Proteomics database No Characterization: Lipid analysis Yes Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 0-750 EV concentration Yes Particle yield Not reported NA EM EM-type Transmission-EM Image type Wide-field

	EV210139	5/6	Homo sapiens	Bone marrow-derived mesenchymal stem cells	(d)(U)C Filtration	Haraszti, Reka A	2016	44%
Study summary Full title High-resolution proteomic and lipidomic analysis of exosomes and microvesicles from different cell sources All authors Reka A Haraszti, Marie-Cecile Didiot, Ellen Sapp, John Leszyk, Scott A Shaffer, Hannah E Rockwell, Fei Gao, Niven R Narain, Marian DiFiglia, Michael A Kiebish, Neil Aronin, Anastasia Khvorova Journal J Extracell Vesicles Abstract Extracellular vesicles (EVs), including exosomes and microvesicles (MVs), are explored for use in di (show more...)Extracellular vesicles (EVs), including exosomes and microvesicles (MVs), are explored for use in diagnostics, therapeutics and drug delivery. However, little is known about the relationship of protein and lipid composition of EVs and their source cells. Here, we report high-resolution lipidomic and proteomic analyses of exosomes and MVs derived by differential ultracentrifugation from 3 different cell types: U87 glioblastoma cells, Huh7 hepatocellular carcinoma cells and human bone marrow-derived mesenchymal stem cells (MSCs). We identified 3,532 proteins and 1,961 lipid species in the screen. Exosomes differed from MVs in several different areas: (a) The protein patterns of exosomes were more likely different from their cells of origin than were the protein patterns of MVs; (b) The proteomes of U87 and Huh7 exosomes were similar to each other but different from the proteomes of MSC exosomes, whereas the lipidomes of Huh7 and MSC exosomes were similar to each other but different from the lipidomes of U87 exosomes; (c) exosomes exhibited proteins of extracellular matrix, heparin-binding, receptors, immune response and cell adhesion functions, whereas MVs were enriched in endoplasmic reticulum, proteasome and mitochondrial proteins. Exosomes and MVs also differed in their types of lipid contents. Enrichment in glycolipids and free fatty acids characterized exosomes, whereas enrichment in ceramides and sphingomyelins characterized MVs. Furthermore, Huh7 and MSC exosomes were specifically enriched in cardiolipins; U87 exosomes were enriched in sphingomyelins. This study comprehensively analyses the protein and lipid composition of exosomes, MVs and source cells in 3 different cell types. (hide) EV-METRIC 44% (84th 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 (shedding) microvesicle 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: CD81/ TSG101/ CD63/ CD9 non-EV: Calnexin Proteomics yes Show all info Study aim Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Bone marrow-derived mesenchymal 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 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 Not specified Pelleting: speed (g) 10000 Filtration steps 0.22µm or 0.2µmNo Characterization: Protein analysis Protein Concentration Method Bradford Western Blot Detected EV-associated proteins CD9/ CD63/ CD81 Not detected EV-associated proteins Tsg101 Not detected contaminants Calnexin Proteomics database No Characterization: Lipid analysis Yes Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 0-500 EV concentration Yes Particle yield Not reported NA EM EM-type Transmission-EM Image type Wide-field

	EV210139	6/6	Homo sapiens	Bone marrow-derived mesenchymal stem cells	(d)(U)C	Haraszti, Reka A	2016	44%
Study summary Full title High-resolution proteomic and lipidomic analysis of exosomes and microvesicles from different cell sources All authors Reka A Haraszti, Marie-Cecile Didiot, Ellen Sapp, John Leszyk, Scott A Shaffer, Hannah E Rockwell, Fei Gao, Niven R Narain, Marian DiFiglia, Michael A Kiebish, Neil Aronin, Anastasia Khvorova Journal J Extracell Vesicles Abstract Extracellular vesicles (EVs), including exosomes and microvesicles (MVs), are explored for use in di (show more...)Extracellular vesicles (EVs), including exosomes and microvesicles (MVs), are explored for use in diagnostics, therapeutics and drug delivery. However, little is known about the relationship of protein and lipid composition of EVs and their source cells. Here, we report high-resolution lipidomic and proteomic analyses of exosomes and MVs derived by differential ultracentrifugation from 3 different cell types: U87 glioblastoma cells, Huh7 hepatocellular carcinoma cells and human bone marrow-derived mesenchymal stem cells (MSCs). We identified 3,532 proteins and 1,961 lipid species in the screen. Exosomes differed from MVs in several different areas: (a) The protein patterns of exosomes were more likely different from their cells of origin than were the protein patterns of MVs; (b) The proteomes of U87 and Huh7 exosomes were similar to each other but different from the proteomes of MSC exosomes, whereas the lipidomes of Huh7 and MSC exosomes were similar to each other but different from the lipidomes of U87 exosomes; (c) exosomes exhibited proteins of extracellular matrix, heparin-binding, receptors, immune response and cell adhesion functions, whereas MVs were enriched in endoplasmic reticulum, proteasome and mitochondrial proteins. Exosomes and MVs also differed in their types of lipid contents. Enrichment in glycolipids and free fatty acids characterized exosomes, whereas enrichment in ceramides and sphingomyelins characterized MVs. Furthermore, Huh7 and MSC exosomes were specifically enriched in cardiolipins; U87 exosomes were enriched in sphingomyelins. This study comprehensively analyses the protein and lipid composition of exosomes, MVs and source cells in 3 different cell types. (hide) EV-METRIC 44% (84th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles exosome Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Protein markers EV: CD81/ TSG101/ CD63/ CD9 non-EV: Calnexin Proteomics yes Show all info Study aim Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Bone marrow-derived mesenchymal 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 10,000 g and 50,000 g Pelleting performed Yes Pelleting: time(min) 30 Pelleting: rotor type Not specified Pelleting: speed (g) 10000 Characterization: Protein analysis Protein Concentration Method Bradford Western Blot Detected EV-associated proteins CD9/ CD63/ CD81 Not detected EV-associated proteins Tsg101 Detected contaminants Calnexin Proteomics database No Characterization: Lipid analysis Yes Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 0-750 EV concentration Yes Particle yield Not reported EM EM-type Transmission-EM Image type Wide-field

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EV-TRACK ID	EV210139
species	Homo sapiens
sample type	Cell culture
cell type	U87	U87	Huh7	Huh7	Bone marrow-derived mesenchymal stem cells	Bone marrow-derived mesenchymal stem cells
condition	Control condition	Control condition	Control condition	Control condition	Control condition	Control condition
separation protocol	(d)(U)C Filtration	(d)(U)C	(d)(U)C Filtration	(d)(U)C	(d)(U)C Filtration	(d)(U)C
vesicle related term	exosome	(shedding) microvesicle	(shedding) microvesicle	exosome	(shedding) microvesicle	exosome
Exp. nr.	1	2	3	4	5	6
EV-METRIC %	44	44	44	44	44	44