EV-TRACK

Search > Results

You searched for: 2020 (Year of publication)

Showing 201 - 250 of 1113

Results list

Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV200117	3/6	Homo sapiens	HEK	(d)(U)C UF qEV	Cocozza, Federico	2020	50%
Study summary Full title Extracellular vesicles containing ACE2 efficiently prevent infection by SARS‐CoV‐2 Spike protein‐containing virus All authors Federico Cocozza, Nathalie Névo, Ester Piovesana, Xavier Lahaye, Julian Buchrieser, Olivier Schwartz, Nicolas Manel, Mercedes Tkach, Clotilde Théry, Lorena Martin‐Jaular Journal J Extracell Vesicles Abstract SARS‐CoV‐2 entry is mediated by binding of the spike protein (S) to the surface receptor ACE2 an (show more...)SARS‐CoV‐2 entry is mediated by binding of the spike protein (S) to the surface receptor ACE2 and subsequent priming by host TMPRSS2 allowing membrane fusion. Here, we produced extracellular vesicles (EVs) exposing ACE2 and demonstrate that ACE2‐EVs are efficient decoys for SARS‐CoV‐2 S protein‐containing lentivirus. Reduction of infectivity positively correlates with the level of ACE2, is much more efficient than with soluble ACE2 and further enhanced by the inclusion of TMPRSS2. (hide) EV-METRIC 50% (87th 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 HEK ACE2 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 Ultrafiltration Commercial method Protein markers EV: CD81/ ACE2/ ADAM10/ CD63/ syntenin-1/ HSP70 non-EV: AChe Proteomics no Show all info Study aim Function/New methodological development Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells HEK EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g Cell viability (%) 89 Cell count 3.5E7-8.5E7 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) 10 Membrane type Regenerated cellulose Commercial kit qEV Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD63/ syntenin-1/ ACE2/ ADAM10/ CD81/ HSP70 Not detected contaminants AChe ELISA Antibody details provided? No Detected EV-associated proteins ACE2 Flow cytometry Antibody details provided? No Detected EV-associated proteins CD63/ CD81/ syntenin-1 Detected EV-associated proteins CD81/ CD63/ syntenin-1 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Median Reported size (nm) 145.5 EV concentration Yes Particle yield Yes, as number of particles per million cells 7.00E+08

	EV200117	4/6	Homo sapiens	HEK	(d)(U)C UF qEV	Cocozza, Federico	2020	50%
Study summary Full title Extracellular vesicles containing ACE2 efficiently prevent infection by SARS‐CoV‐2 Spike protein‐containing virus All authors Federico Cocozza, Nathalie Névo, Ester Piovesana, Xavier Lahaye, Julian Buchrieser, Olivier Schwartz, Nicolas Manel, Mercedes Tkach, Clotilde Théry, Lorena Martin‐Jaular Journal J Extracell Vesicles Abstract SARS‐CoV‐2 entry is mediated by binding of the spike protein (S) to the surface receptor ACE2 an (show more...)SARS‐CoV‐2 entry is mediated by binding of the spike protein (S) to the surface receptor ACE2 and subsequent priming by host TMPRSS2 allowing membrane fusion. Here, we produced extracellular vesicles (EVs) exposing ACE2 and demonstrate that ACE2‐EVs are efficient decoys for SARS‐CoV‐2 S protein‐containing lentivirus. Reduction of infectivity positively correlates with the level of ACE2, is much more efficient than with soluble ACE2 and further enhanced by the inclusion of TMPRSS2. (hide) EV-METRIC 50% (87th 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 HEK ACE2 and TMPRSS2 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 Ultrafiltration Commercial method Protein markers EV: CD81/ ACE2/ ADAM10/ CD63/ syntenin-1/ HSP70 non-EV: AChe Proteomics no Show all info Study aim Function/New methodological development Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells HEK EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g Cell viability (%) 89 Cell count 3.5E7-8.5E7 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) 10 Membrane type Regenerated cellulose Commercial kit qEV Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins syntenin-1/ ACE2/ ADAM10/ CD63/ CD81/ HSP70 Not detected contaminants AChe ELISA Antibody details provided? No Detected EV-associated proteins ACE2 Flow cytometry Antibody details provided? No Detected EV-associated proteins CD63/ CD81/ syntenin-1 Detected EV-associated proteins CD81/ CD63/ syntenin-1 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Median Reported size (nm) 153.9 EV concentration Yes Particle yield Yes, as number of particles per million cells 6.00E+08

	EV200068	1/5	Homo sapiens	Blood plasma	(d)(U)C SEC (non-commercial) Filtration	Linda Hofmann	2020	50%
Study summary Full title The Potential of CD16 on Plasma-Derived Exosomes as a Liquid Biomarker in Head and Neck Cancer All authors Linda Hofmann, Sonja Ludwig, Patrick J Schuler, Thomas K Hoffmann, Cornelia Brunner, Marie-Nicole Theodoraki Journal Int J Mol Sci Abstract Head and neck squamous cell carcinomas (HNSCC) are highly immune suppressive and aggressive malignan (show more...)Head and neck squamous cell carcinomas (HNSCC) are highly immune suppressive and aggressive malignancies. As part of the tumor microenvironment, exosomes contribute to this immune suppression. The Fc receptor CD16 is widely expressed on monocytes, neutrophils, and natural killer (NK) cells and is involved in antibody-dependent cell-mediated cytotoxicity (ADCC). Here, surface levels of CD16 on total exosomes and tumor-derived exosomes (TEX) from plasma of HNSCC patients were analyzed regarding their potential as liquid biomarkers for disease stage. Exosomes were isolated from plasma using mini size exclusion chromatography. TEX were enriched by immune affinity capture with CD44v3 antibodies. On-bead flow cytometry was used to measure CD16 levels on total exosomes and TEX. The results were correlated with clinicopathological parameters. Total exosomes from HNSCC patients had significantly higher CD16 levels compared to TEX. Further, CD16 surface levels of total exosomes, but not TEX, correlated with clinicopathological parameters. Patients with advanced tumor stages T3/4 and Union for International Cancer Control (UICC) stages III/IV had significantly higher CD16 levels on total exosomes compared to patients with early tumor stages T1/2 and UICC stages I/II, respectively. Overall, CD16 positive exosomes have the potential as liquid biomarkers for HNSCC tumor stage and aggressiveness. (hide) EV-METRIC 50% (83rd 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 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 SEC (non-commercial) Filtration Protein markers EV: TSG101/ CD81/ CD63/ CD9/ CD16 non-EV: Grp94/ ApoA1 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 Pelleting performed No Filtration steps 0.22µm or 0.2µm Size-exclusion chromatography Total column volume (mL) 10 Sample volume/column (mL) 1 Resin type Sepharose CL-2B Other Name other separation method SEC (non-commercial) 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/ CD81 Not detected contaminants ApoA1/ Grp94 Flow cytometry aspecific beads Antibody details provided? No Detected EV-associated proteins CD16 Flow cytometry specific beads Antibody details provided? No Antibody dilution provided? No Selected surface protein(s) CD44v3 Detected EV-associated proteins CD16 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 30-150 EM EM-type Transmission-EM Image type Wide-field

	EV200068	2/5	Homo sapiens	Blood plasma	(d)(U)C SEC (non-commercial) Filtration	Linda Hofmann	2020	50%
Study summary Full title The Potential of CD16 on Plasma-Derived Exosomes as a Liquid Biomarker in Head and Neck Cancer All authors Linda Hofmann, Sonja Ludwig, Patrick J Schuler, Thomas K Hoffmann, Cornelia Brunner, Marie-Nicole Theodoraki Journal Int J Mol Sci Abstract Head and neck squamous cell carcinomas (HNSCC) are highly immune suppressive and aggressive malignan (show more...)Head and neck squamous cell carcinomas (HNSCC) are highly immune suppressive and aggressive malignancies. As part of the tumor microenvironment, exosomes contribute to this immune suppression. The Fc receptor CD16 is widely expressed on monocytes, neutrophils, and natural killer (NK) cells and is involved in antibody-dependent cell-mediated cytotoxicity (ADCC). Here, surface levels of CD16 on total exosomes and tumor-derived exosomes (TEX) from plasma of HNSCC patients were analyzed regarding their potential as liquid biomarkers for disease stage. Exosomes were isolated from plasma using mini size exclusion chromatography. TEX were enriched by immune affinity capture with CD44v3 antibodies. On-bead flow cytometry was used to measure CD16 levels on total exosomes and TEX. The results were correlated with clinicopathological parameters. Total exosomes from HNSCC patients had significantly higher CD16 levels compared to TEX. Further, CD16 surface levels of total exosomes, but not TEX, correlated with clinicopathological parameters. Patients with advanced tumor stages T3/4 and Union for International Cancer Control (UICC) stages III/IV had significantly higher CD16 levels on total exosomes compared to patients with early tumor stages T1/2 and UICC stages I/II, respectively. Overall, CD16 positive exosomes have the potential as liquid biomarkers for HNSCC tumor stage and aggressiveness. (hide) EV-METRIC 50% (83rd 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 HNSCC 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 SEC (non-commercial) Filtration Protein markers EV: TSG101/ CD81/ CD63/ CD9/ CD16 non-EV: Grp94/ ApoA1 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 Pelleting performed No Filtration steps 0.22µm or 0.2µm Size-exclusion chromatography Total column volume (mL) 10 Sample volume/column (mL) 1 Resin type Sepharose CL-2B Other Name other separation method SEC (non-commercial) 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/ CD81 Not detected contaminants ApoA1/ Grp94 Flow cytometry aspecific beads Antibody details provided? No Detected EV-associated proteins CD16 Flow cytometry specific beads Antibody details provided? No Antibody dilution provided? No Selected surface protein(s) CD44v3 Detected EV-associated proteins CD16 Characterization: Lipid analysis No

	EV200063	1/2	Homo sapiens	Blood plasma	qEV	Silvia Picciolini	2020	50%
Study summary Full title An SPRi-based biosensor pilot study: Analysis of multiple circulating extracellular vesicles and hippocampal volume in Alzheimer’s disease All authors Silvia Picciolini,Alice Gualerzi,Cristiano Carlomagno,Monia Cabinio,Stefano Sorrentino,Francesca Baglio,Marzia Bedoni Journal ACS Nano Abstract One of the main hurdles in the study of Alzheimer’s Disease (AD) is the lack of easily accessible (show more...)One of the main hurdles in the study of Alzheimer’s Disease (AD) is the lack of easily accessible and sensitive biomarkers for the diagnosis, the prediction of the disease progression rate and the evaluation of rehabilitative and pharmacological treatments. Extracellular Vesicles (EVs) are nanoscales particles released by body cells studied as promising biomarkers of AD as they are involved in the onset and progression of the disease. In the strive for a reliable and sensitive method to analyze EVs, we applied our recently developed biosensor based on Surface Plasmon Resonance imaging (SPRi) technology for the identification and profiling of neural EVs populations circulating in the plasma of 10 AD patients and 10 healthy subjects. The SPRi-array was designed to separate simultaneously EVs released by neurons, astrocytes, microglia and oligodendrocytes, and to evaluate the presence and the relative amount of specific surface molecules related to pathological processes including translocator protein (TSPO), β-Amyloid and ganglioside M1. (hide) EV-METRIC 50% (83rd 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 qEV Protein markers EV: CD9/ CD171/ Glast/ PLP1/ CD11b/ EphrinB non-EV: Ig Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Blood plasma Separation Method Commercial kit qEV Other Name other separation method qEV Characterization: Protein analysis Protein Concentration Method BCA Detected EV-associated proteins CD9/ CD171/ Glast/ PLP1/ CD11b/ EphrinB Not detected contaminants Ig Characterization: Lipid analysis Yes Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 166,45 EV concentration Yes EM EM-type Transmission-EM Image type Close-up

	EV200063	2/2	Homo sapiens	Blood plasma	qEV	Silvia Picciolini	2020	50%
Study summary Full title An SPRi-based biosensor pilot study: Analysis of multiple circulating extracellular vesicles and hippocampal volume in Alzheimer’s disease All authors Silvia Picciolini,Alice Gualerzi,Cristiano Carlomagno,Monia Cabinio,Stefano Sorrentino,Francesca Baglio,Marzia Bedoni Journal ACS Nano Abstract One of the main hurdles in the study of Alzheimer’s Disease (AD) is the lack of easily accessible (show more...)One of the main hurdles in the study of Alzheimer’s Disease (AD) is the lack of easily accessible and sensitive biomarkers for the diagnosis, the prediction of the disease progression rate and the evaluation of rehabilitative and pharmacological treatments. Extracellular Vesicles (EVs) are nanoscales particles released by body cells studied as promising biomarkers of AD as they are involved in the onset and progression of the disease. In the strive for a reliable and sensitive method to analyze EVs, we applied our recently developed biosensor based on Surface Plasmon Resonance imaging (SPRi) technology for the identification and profiling of neural EVs populations circulating in the plasma of 10 AD patients and 10 healthy subjects. The SPRi-array was designed to separate simultaneously EVs released by neurons, astrocytes, microglia and oligodendrocytes, and to evaluate the presence and the relative amount of specific surface molecules related to pathological processes including translocator protein (TSPO), β-Amyloid and ganglioside M1. (hide) EV-METRIC 50% (83rd 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 Alzheimer disease 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 Protein markers EV: CD9/ CD171/ Glast/ PLP1/ CD11b/ EphrinB non-EV: Ig Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Blood plasma Separation Method Commercial kit qEV Other Name other separation method qEV Characterization: Protein analysis Protein Concentration Method BCA Detected EV-associated proteins CD9/ CD171/ Glast/ PLP1/ CD11b/ EphrinB Not detected contaminants Ig Characterization: Lipid analysis Yes Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 183,28 EV concentration Yes EM EM-type Transmission-EM Image type Close-up

	EV200060	1/4	Mus musculus	Control condition	(d)(U)C ExoQuick	de Mendonça, Mariana	2020	50%
Study summary Full title Aerobic exercise training regulates serum extracellular vesicle miRNAs linked to obesity to promote their beneficial effects in mice All authors Mariana de Mendonça, Karina C Rocha, Érica de Sousa, Beatriz M V Pereira, Lila Missae Oyama, Alice C Rodrigues Journal Am J Physiol Endocrinol Metab Abstract There is a growing body of evidence that extracellular vesicles (EVs) and their cargo of RNA, DNA, a (show more...)There is a growing body of evidence that extracellular vesicles (EVs) and their cargo of RNA, DNA, and protein are released in the circulation with exercise and might mediate interorgan communication. C57BL6/J male mice were subjected to diet-induced obesity and aerobic training on a treadmill for 8 wk. The effect of aerobic training was evaluated in the liver, muscle, kidney, and white/brown adipose tissue. To provide new mechanistic insight, we profiled miRNA from serum EVs of obese and obese trained mice. We demonstrate that aerobic training changes the circulating EV miRNA profile of obese mice, including decreases in miR-122, miR-192, and miR-22 levels. Circulating miRNA levels were associated with miRNA levels in mouse liver white adipose tissue (WAT). In WAT, aerobically trained obese mice showed reduced adipocyte hypertrophy and increased the number of smaller adipocytes and the expression of Cebpa, Pparg, Fabp4 (adipogenesis markers), and ATP-citrate lyase enzyme activity. Importantly, miR-22 levels negatively correlated with the expression of adipogenesis and insulin sensitivity markers. In the liver, aerobic training reverted obesity-induced steatohepatitis, and steatosis score and Pparg expression were negatively correlated with miR-122 levels. The prometabolic effects of aerobic exercise in obesity possibly involve EV miRNAs, which might be involved in communication between liver and WAT. Our data provide significant evidence demonstrating that aerobic training exercise-induced EVs mediate the effect of exercise on adipose tissue metabolism. (hide) EV-METRIC 50% (50th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Control condition Sample origin No extra separation steps 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 Commercial method Protein markers EV: TSG101/ Alix/ CD63/ CD9 non-EV: Grp94/ Albumin Proteomics no Show all info Study aim Biomarker Sample Species Mus musculus Sample Type Control condition Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Pelleting performed No Commercial kit ExoQuick 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/ TSG101/ Alix Detected contaminants Albumin Not detected contaminants Grp94 Characterization: RNA analysis RNA analysis Type (RT)(q)PCR;RNA sequencing Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis DLS Report type Mean Reported size (nm) 110 Used for determining EV concentration? Yes NTA Report type Size range/distribution Reported size (nm) 164 EV concentration Yes

	EV200060	3/4	Mus musculus	high-fat diet	(d)(U)C ExoQuick	de Mendonça, Mariana	2020	50%
Study summary Full title Aerobic exercise training regulates serum extracellular vesicle miRNAs linked to obesity to promote their beneficial effects in mice All authors Mariana de Mendonça, Karina C Rocha, Érica de Sousa, Beatriz M V Pereira, Lila Missae Oyama, Alice C Rodrigues Journal Am J Physiol Endocrinol Metab Abstract There is a growing body of evidence that extracellular vesicles (EVs) and their cargo of RNA, DNA, a (show more...)There is a growing body of evidence that extracellular vesicles (EVs) and their cargo of RNA, DNA, and protein are released in the circulation with exercise and might mediate interorgan communication. C57BL6/J male mice were subjected to diet-induced obesity and aerobic training on a treadmill for 8 wk. The effect of aerobic training was evaluated in the liver, muscle, kidney, and white/brown adipose tissue. To provide new mechanistic insight, we profiled miRNA from serum EVs of obese and obese trained mice. We demonstrate that aerobic training changes the circulating EV miRNA profile of obese mice, including decreases in miR-122, miR-192, and miR-22 levels. Circulating miRNA levels were associated with miRNA levels in mouse liver white adipose tissue (WAT). In WAT, aerobically trained obese mice showed reduced adipocyte hypertrophy and increased the number of smaller adipocytes and the expression of Cebpa, Pparg, Fabp4 (adipogenesis markers), and ATP-citrate lyase enzyme activity. Importantly, miR-22 levels negatively correlated with the expression of adipogenesis and insulin sensitivity markers. In the liver, aerobic training reverted obesity-induced steatohepatitis, and steatosis score and Pparg expression were negatively correlated with miR-122 levels. The prometabolic effects of aerobic exercise in obesity possibly involve EV miRNAs, which might be involved in communication between liver and WAT. Our data provide significant evidence demonstrating that aerobic training exercise-induced EVs mediate the effect of exercise on adipose tissue metabolism. (hide) EV-METRIC 50% (50th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type high-fat diet Sample origin No extra separation steps 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 Commercial method Protein markers EV: Alix/ TSG101/ CD63/ CD9 non-EV: Grp94/ Albumin Proteomics no Show all info Study aim Biomarker Sample Species Mus musculus Sample Type high-fat diet Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Pelleting performed No Commercial kit ExoQuick 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/ TSG101 Detected contaminants Albumin Not detected contaminants Grp94 Characterization: RNA analysis RNA analysis Type (RT)(q)PCR;RNAsequencing Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis DLS Report type Not Reported NTA Report type Not Reported

	EV200051	2/2	Homo sapiens	Serum	IAF (d)(U)C Filtration qEV	Tzaridis, Theophilos	2020	50%
Study summary Full title Analysis of Serum miRNA in Glioblastoma Patients: CD44-Based Enrichment of Extracellular Vesicles Enhances Specificity for the Prognostic Signature All authors Theophilos Tzaridis, Katrin S Reiners, Johannes Weller, Daniel Bachurski, Niklas Schäfer, Christina Schaub, Michael Hallek, Björn Scheffler, Martin Glas, Ulrich Herrlinger, Stefan Wild, Christoph Coch, Gunther Hartmann Journal Int J Mol Sci Abstract Glioblastoma is a devastating disease, for which biomarkers allowing a prediction of prognosis are u (show more...)Glioblastoma is a devastating disease, for which biomarkers allowing a prediction of prognosis are urgently needed. microRNAs have been described as potentially valuable biomarkers in cancer. Here, we studied a panel of microRNAs in extracellular vesicles (EVs) from the serum of glioblastoma patients and evaluated their correlation with the prognosis of these patients. The levels of 15 microRNAs in EVs that were separated by size-exclusion chromatography were studied by quantitative real-time PCR, followed by CD44 immunoprecipitation (SEC + CD44), and compared with those from the total serum of glioblastoma patients (n = 55) and healthy volunteers (n = 10). Compared to total serum, we found evidence for the enrichment of miR-21-3p and miR-106a-5p and, conversely, lower levels of miR-15b-3p, in SEC + CD44 EVs. miR-15b-3p and miR-21-3p were upregulated in glioblastoma patients compared to healthy subjects. A significant correlation with survival of the patients was found for levels of miR-15b-3p in total serum and miR-15b-3p, miR-21-3p, miR-106a-5p, and miR-328-3p in SEC + CD44 EVs. Combining miR-15b-3p in serum or miR-106a-5p in SEC + CD44 EVs with any one of the other three microRNAs in SEC + CD44 EVs allowed for a prognostic stratification of glioblastoma patients. We have thus identified four microRNAs in glioblastoma patients whose levels, in combination, can predict the prognosis for these patients. (hide) EV-METRIC 50% (89th 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 glioblastoma multiforme 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 Immunoaffinity capture (non-commercial) (d)(U)C Filtration Commercial method Protein markers EV: Flotillin1/ CD9 non-EV: Calnexin Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Serum Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Pelleting performed No Filtration steps 0.45µm > x > 0.22µm, Commercial kit qEV Immunoaffinity capture Selected surface protein(s) CD44 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 Flotillin1/ CD9 Not detected contaminants Calnexin Characterization: RNA analysis RNA analysis Type (RT)(q)PCR Database Yes Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Median Reported size (nm) 115 +/- 22 EV concentration Yes Particle yield number of particles/ml with constant reconstitution volume 1.7E+12+/- 9.1E+12 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV200008	2/18	Homo sapiens	Blood plasma	Wayen Exosome Isolation kit	Peng, Cheng	2020	50%
Study summary Full title Isolation of extracellular vesicle with different precipitation-based methods exerts a tremendous impact on the biomarker analysis for clinical plasma samples All authors Cheng Peng, Jizhuang Wang, Qiyuan Bao, Jun Wang, Zhuochao Liu, Junxiang Wen, Weibin Zhang, Yuhui Shen Journal Cancer Biomarkers Abstract Background: Extracellular vesicles(EVs) is an emerging approach of cancer liquid biopsy. Although th (show more...)Background: Extracellular vesicles(EVs) is an emerging approach of cancer liquid biopsy. Although the precipitation-based method with commercial kits has gained popularity as the second most commonly used technique, these protocols vary tremendously with many included reagents still unknown to the community. Methods: In this study, we assigned each of the 3 clinical plasma samples into 6 aliquots to assess five commercial EV isolation kits, in comparison with ultracentrifugation(UC). We implemented a standardized EV preparation and transcriptome analysis workflow except the EV isolation methods used. The metrics of EVs and its RNA cargo (evRNA) were compared to assess the technical variations versus the biological variations in the clinical setting. Results: Although the size range of the isolated EVs demonstrated a similar distribution, we found significant technical variability among these methods, in terms of EV amount, purity, subpopulations and RNA integrity. Such variabilities were further relayed to a drastic divergence of evRNA expression on a transcriptome-wide fashion. Conclusions: Our study demonstrated a highly variable result from polymeric precipitation-based EV isolation methods, making EVs based biomarker analysis difficult to interpret and reproduce. We highlighted the importance of benchmarking and transparent reporting of the precipitation-based protocols in the liquid biopsy research. (hide) EV-METRIC 50% (83rd 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 Wayen Exosome Isolation kit Protein markers EV: TSG101/ CD81/ CD63/ CD9/ vimentin non-EV: calnexin/ Albumin/ Apolipoprotein A-1 Proteomics no Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Blood plasma Separation Method Commercial kit Other;Wayen Exosome Isolation kit Other Name other separation method Wayen Exosome Isolation kit 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/ vimentin/ TSG101/ CD81 Detected contaminants Apolipoprotein A-1/ Albumin Not detected contaminants calnexin Characterization: RNA analysis RNA analysis Type RNAsequencing Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 30-240 EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 86700000000

	EV200008	3/18	Homo sapiens	Blood plasma	ExoQuick	Peng, Cheng	2020	50%
Study summary Full title Isolation of extracellular vesicle with different precipitation-based methods exerts a tremendous impact on the biomarker analysis for clinical plasma samples All authors Cheng Peng, Jizhuang Wang, Qiyuan Bao, Jun Wang, Zhuochao Liu, Junxiang Wen, Weibin Zhang, Yuhui Shen Journal Cancer Biomarkers Abstract Background: Extracellular vesicles(EVs) is an emerging approach of cancer liquid biopsy. Although th (show more...)Background: Extracellular vesicles(EVs) is an emerging approach of cancer liquid biopsy. Although the precipitation-based method with commercial kits has gained popularity as the second most commonly used technique, these protocols vary tremendously with many included reagents still unknown to the community. Methods: In this study, we assigned each of the 3 clinical plasma samples into 6 aliquots to assess five commercial EV isolation kits, in comparison with ultracentrifugation(UC). We implemented a standardized EV preparation and transcriptome analysis workflow except the EV isolation methods used. The metrics of EVs and its RNA cargo (evRNA) were compared to assess the technical variations versus the biological variations in the clinical setting. Results: Although the size range of the isolated EVs demonstrated a similar distribution, we found significant technical variability among these methods, in terms of EV amount, purity, subpopulations and RNA integrity. Such variabilities were further relayed to a drastic divergence of evRNA expression on a transcriptome-wide fashion. Conclusions: Our study demonstrated a highly variable result from polymeric precipitation-based EV isolation methods, making EVs based biomarker analysis difficult to interpret and reproduce. We highlighted the importance of benchmarking and transparent reporting of the precipitation-based protocols in the liquid biopsy research. (hide) EV-METRIC 50% (83rd 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 ExoQuick Protein markers EV: TSG101/ CD81/ CD63/ CD9/ vimentin non-EV: calnexin/ Albumin/ Apolipoprotein A-1 Proteomics no Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Blood plasma Separation Method Commercial kit ExoQuick Other Name other separation method ExoQuick 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/ vimentin/ CD81 Detected contaminants Apolipoprotein A-1/ Albumin Not detected contaminants calnexin Characterization: RNA analysis RNA analysis Type RNAsequencing Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 30-240 EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 24300000000

	EV200008	4/18	Homo sapiens	Blood plasma	(d)(U)C	Peng, Cheng	2020	50%
Study summary Full title Isolation of extracellular vesicle with different precipitation-based methods exerts a tremendous impact on the biomarker analysis for clinical plasma samples All authors Cheng Peng, Jizhuang Wang, Qiyuan Bao, Jun Wang, Zhuochao Liu, Junxiang Wen, Weibin Zhang, Yuhui Shen Journal Cancer Biomarkers Abstract Background: Extracellular vesicles(EVs) is an emerging approach of cancer liquid biopsy. Although th (show more...)Background: Extracellular vesicles(EVs) is an emerging approach of cancer liquid biopsy. Although the precipitation-based method with commercial kits has gained popularity as the second most commonly used technique, these protocols vary tremendously with many included reagents still unknown to the community. Methods: In this study, we assigned each of the 3 clinical plasma samples into 6 aliquots to assess five commercial EV isolation kits, in comparison with ultracentrifugation(UC). We implemented a standardized EV preparation and transcriptome analysis workflow except the EV isolation methods used. The metrics of EVs and its RNA cargo (evRNA) were compared to assess the technical variations versus the biological variations in the clinical setting. Results: Although the size range of the isolated EVs demonstrated a similar distribution, we found significant technical variability among these methods, in terms of EV amount, purity, subpopulations and RNA integrity. Such variabilities were further relayed to a drastic divergence of evRNA expression on a transcriptome-wide fashion. Conclusions: Our study demonstrated a highly variable result from polymeric precipitation-based EV isolation methods, making EVs based biomarker analysis difficult to interpret and reproduce. We highlighted the importance of benchmarking and transparent reporting of the precipitation-based protocols in the liquid biopsy research. (hide) EV-METRIC 50% (83rd 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 primary osteosarcoma Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Protein markers EV: TSG101/ CD81/ CD63/ CD9/ vimentin non-EV: calnexin/ Albumin/ Apolipoprotein A-1 Proteomics no Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Blood plasma 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) 660 Pelleting: rotor type Type 50.2 Ti Pelleting: speed (g) 110000 Wash: volume per pellet (ml) 20 Wash: time (min) 90 Wash: Rotor Type Type 50.2 Ti Wash: speed (g) 110000 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 vimentin/ CD9/ CD63/ TSG101/ CD81 Detected contaminants Apolipoprotein A-1/ Albumin Not detected contaminants calnexin Characterization: RNA analysis RNA analysis Type RNAsequencing Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 30-240 EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 733000000

	EV200008	5/18	Homo sapiens	Blood plasma	Wayen Exosome Isolation kit	Peng, Cheng	2020	50%
Study summary Full title Isolation of extracellular vesicle with different precipitation-based methods exerts a tremendous impact on the biomarker analysis for clinical plasma samples All authors Cheng Peng, Jizhuang Wang, Qiyuan Bao, Jun Wang, Zhuochao Liu, Junxiang Wen, Weibin Zhang, Yuhui Shen Journal Cancer Biomarkers Abstract Background: Extracellular vesicles(EVs) is an emerging approach of cancer liquid biopsy. Although th (show more...)Background: Extracellular vesicles(EVs) is an emerging approach of cancer liquid biopsy. Although the precipitation-based method with commercial kits has gained popularity as the second most commonly used technique, these protocols vary tremendously with many included reagents still unknown to the community. Methods: In this study, we assigned each of the 3 clinical plasma samples into 6 aliquots to assess five commercial EV isolation kits, in comparison with ultracentrifugation(UC). We implemented a standardized EV preparation and transcriptome analysis workflow except the EV isolation methods used. The metrics of EVs and its RNA cargo (evRNA) were compared to assess the technical variations versus the biological variations in the clinical setting. Results: Although the size range of the isolated EVs demonstrated a similar distribution, we found significant technical variability among these methods, in terms of EV amount, purity, subpopulations and RNA integrity. Such variabilities were further relayed to a drastic divergence of evRNA expression on a transcriptome-wide fashion. Conclusions: Our study demonstrated a highly variable result from polymeric precipitation-based EV isolation methods, making EVs based biomarker analysis difficult to interpret and reproduce. We highlighted the importance of benchmarking and transparent reporting of the precipitation-based protocols in the liquid biopsy research. (hide) EV-METRIC 50% (83rd 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 primary osteosarcoma 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 Wayen Exosome Isolation kit Protein markers EV: TSG101/ CD81/ CD63/ CD9/ vimentin non-EV: calnexin/ Albumin/ Apolipoprotein A-1 Proteomics no Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Blood plasma Separation Method Commercial kit Other;Wayen Exosome Isolation kit Other Name other separation method Wayen Exosome Isolation kit 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 vimentin/ CD9/ CD63/ TSG101/ CD81 Detected contaminants Apolipoprotein A-1/ Albumin Not detected contaminants calnexin Characterization: RNA analysis RNA analysis Type RNAsequencing Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 30-240 EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 90000000000

	EV200008	6/18	Homo sapiens	Blood plasma	ExoQuick	Peng, Cheng	2020	50%
Study summary Full title Isolation of extracellular vesicle with different precipitation-based methods exerts a tremendous impact on the biomarker analysis for clinical plasma samples All authors Cheng Peng, Jizhuang Wang, Qiyuan Bao, Jun Wang, Zhuochao Liu, Junxiang Wen, Weibin Zhang, Yuhui Shen Journal Cancer Biomarkers Abstract Background: Extracellular vesicles(EVs) is an emerging approach of cancer liquid biopsy. Although th (show more...)Background: Extracellular vesicles(EVs) is an emerging approach of cancer liquid biopsy. Although the precipitation-based method with commercial kits has gained popularity as the second most commonly used technique, these protocols vary tremendously with many included reagents still unknown to the community. Methods: In this study, we assigned each of the 3 clinical plasma samples into 6 aliquots to assess five commercial EV isolation kits, in comparison with ultracentrifugation(UC). We implemented a standardized EV preparation and transcriptome analysis workflow except the EV isolation methods used. The metrics of EVs and its RNA cargo (evRNA) were compared to assess the technical variations versus the biological variations in the clinical setting. Results: Although the size range of the isolated EVs demonstrated a similar distribution, we found significant technical variability among these methods, in terms of EV amount, purity, subpopulations and RNA integrity. Such variabilities were further relayed to a drastic divergence of evRNA expression on a transcriptome-wide fashion. Conclusions: Our study demonstrated a highly variable result from polymeric precipitation-based EV isolation methods, making EVs based biomarker analysis difficult to interpret and reproduce. We highlighted the importance of benchmarking and transparent reporting of the precipitation-based protocols in the liquid biopsy research. (hide) EV-METRIC 50% (83rd 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 primary osteosarcoma 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 ExoQuick Protein markers EV: TSG101/ CD81/ CD63/ CD9/ vimentin non-EV: calnexin/ Albumin/ Apolipoprotein A-1 Proteomics no Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Blood plasma Separation Method Commercial kit ExoQuick Other Name other separation method ExoQuick 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 vimentin/ TSG101/ CD9/ CD63/ CD81 Detected contaminants Apolipoprotein A-1/ Albumin Not detected contaminants calnexin Characterization: RNA analysis RNA analysis Type RNAsequencing Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 30-240 EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 53300000000

	EV200008	7/18	Homo sapiens	Blood plasma	(d)(U)C	Peng, Cheng	2020	50%
Study summary Full title Isolation of extracellular vesicle with different precipitation-based methods exerts a tremendous impact on the biomarker analysis for clinical plasma samples All authors Cheng Peng, Jizhuang Wang, Qiyuan Bao, Jun Wang, Zhuochao Liu, Junxiang Wen, Weibin Zhang, Yuhui Shen Journal Cancer Biomarkers Abstract Background: Extracellular vesicles(EVs) is an emerging approach of cancer liquid biopsy. Although th (show more...)Background: Extracellular vesicles(EVs) is an emerging approach of cancer liquid biopsy. Although the precipitation-based method with commercial kits has gained popularity as the second most commonly used technique, these protocols vary tremendously with many included reagents still unknown to the community. Methods: In this study, we assigned each of the 3 clinical plasma samples into 6 aliquots to assess five commercial EV isolation kits, in comparison with ultracentrifugation(UC). We implemented a standardized EV preparation and transcriptome analysis workflow except the EV isolation methods used. The metrics of EVs and its RNA cargo (evRNA) were compared to assess the technical variations versus the biological variations in the clinical setting. Results: Although the size range of the isolated EVs demonstrated a similar distribution, we found significant technical variability among these methods, in terms of EV amount, purity, subpopulations and RNA integrity. Such variabilities were further relayed to a drastic divergence of evRNA expression on a transcriptome-wide fashion. Conclusions: Our study demonstrated a highly variable result from polymeric precipitation-based EV isolation methods, making EVs based biomarker analysis difficult to interpret and reproduce. We highlighted the importance of benchmarking and transparent reporting of the precipitation-based protocols in the liquid biopsy research. (hide) EV-METRIC 50% (83rd 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 Metastatic osteosarcoma Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Protein markers EV: TSG101/ CD81/ CD63/ CD9/ vimentin non-EV: calnexin/ Albumin/ Apolipoprotein A-1 Proteomics no Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Blood plasma 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) 660 Pelleting: rotor type Type 50.2 Ti Pelleting: speed (g) 110000 Wash: volume per pellet (ml) 20 Wash: time (min) 90 Wash: Rotor Type Type 50.2 Ti Wash: speed (g) 110000 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 vimentin/ TSG101/ CD9/ CD63/ CD81 Detected contaminants Apolipoprotein A-1/ Albumin Not detected contaminants calnexin Characterization: RNA analysis RNA analysis Type RNAsequencing Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 30-240 EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 6670000000

	EV200008	8/18	Homo sapiens	Blood plasma	Wayen Exosome Isolation kit	Peng, Cheng	2020	50%
Study summary Full title Isolation of extracellular vesicle with different precipitation-based methods exerts a tremendous impact on the biomarker analysis for clinical plasma samples All authors Cheng Peng, Jizhuang Wang, Qiyuan Bao, Jun Wang, Zhuochao Liu, Junxiang Wen, Weibin Zhang, Yuhui Shen Journal Cancer Biomarkers Abstract Background: Extracellular vesicles(EVs) is an emerging approach of cancer liquid biopsy. Although th (show more...)Background: Extracellular vesicles(EVs) is an emerging approach of cancer liquid biopsy. Although the precipitation-based method with commercial kits has gained popularity as the second most commonly used technique, these protocols vary tremendously with many included reagents still unknown to the community. Methods: In this study, we assigned each of the 3 clinical plasma samples into 6 aliquots to assess five commercial EV isolation kits, in comparison with ultracentrifugation(UC). We implemented a standardized EV preparation and transcriptome analysis workflow except the EV isolation methods used. The metrics of EVs and its RNA cargo (evRNA) were compared to assess the technical variations versus the biological variations in the clinical setting. Results: Although the size range of the isolated EVs demonstrated a similar distribution, we found significant technical variability among these methods, in terms of EV amount, purity, subpopulations and RNA integrity. Such variabilities were further relayed to a drastic divergence of evRNA expression on a transcriptome-wide fashion. Conclusions: Our study demonstrated a highly variable result from polymeric precipitation-based EV isolation methods, making EVs based biomarker analysis difficult to interpret and reproduce. We highlighted the importance of benchmarking and transparent reporting of the precipitation-based protocols in the liquid biopsy research. (hide) EV-METRIC 50% (83rd 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 Metastatic osteosarcoma 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 Wayen Exosome Isolation kit Protein markers EV: TSG101/ CD81/ CD63/ CD9/ vimentin non-EV: calnexin/ Albumin/ Apolipoprotein A-1 Proteomics no Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Blood plasma Separation Method Commercial kit Other;Wayen Exosome Isolation kit Other Name other separation method Wayen Exosome Isolation kit 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 vimentin/ CD9/ CD63/ TSG101/ CD81 Detected contaminants Apolipoprotein A-1/ Albumin Not detected contaminants calnexin Characterization: RNA analysis RNA analysis Type RNAsequencing Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 30-240 EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 227000000000

	EV200008	9/18	Homo sapiens	Blood plasma	ExoQuick	Peng, Cheng	2020	50%
Study summary Full title Isolation of extracellular vesicle with different precipitation-based methods exerts a tremendous impact on the biomarker analysis for clinical plasma samples All authors Cheng Peng, Jizhuang Wang, Qiyuan Bao, Jun Wang, Zhuochao Liu, Junxiang Wen, Weibin Zhang, Yuhui Shen Journal Cancer Biomarkers Abstract Background: Extracellular vesicles(EVs) is an emerging approach of cancer liquid biopsy. Although th (show more...)Background: Extracellular vesicles(EVs) is an emerging approach of cancer liquid biopsy. Although the precipitation-based method with commercial kits has gained popularity as the second most commonly used technique, these protocols vary tremendously with many included reagents still unknown to the community. Methods: In this study, we assigned each of the 3 clinical plasma samples into 6 aliquots to assess five commercial EV isolation kits, in comparison with ultracentrifugation(UC). We implemented a standardized EV preparation and transcriptome analysis workflow except the EV isolation methods used. The metrics of EVs and its RNA cargo (evRNA) were compared to assess the technical variations versus the biological variations in the clinical setting. Results: Although the size range of the isolated EVs demonstrated a similar distribution, we found significant technical variability among these methods, in terms of EV amount, purity, subpopulations and RNA integrity. Such variabilities were further relayed to a drastic divergence of evRNA expression on a transcriptome-wide fashion. Conclusions: Our study demonstrated a highly variable result from polymeric precipitation-based EV isolation methods, making EVs based biomarker analysis difficult to interpret and reproduce. We highlighted the importance of benchmarking and transparent reporting of the precipitation-based protocols in the liquid biopsy research. (hide) EV-METRIC 50% (83rd 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 Metastatic osteosarcoma 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 ExoQuick Protein markers EV: TSG101/ CD81/ CD63/ CD9/ vimentin non-EV: calnexin/ Albumin/ Apolipoprotein A-1 Proteomics no Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Blood plasma Separation Method Commercial kit ExoQuick Other Name other separation method ExoQuick 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 vimentin/ TSG101/ CD9/ CD63/ CD81 Detected contaminants Apolipoprotein A-1/ Albumin Not detected contaminants calnexin Characterization: RNA analysis RNA analysis Type RNAsequencing Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 30-240 EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 32000000000

	EV200008	11/18	Homo sapiens	Blood plasma	Ribo Exosome Isolation Reagent	Peng, Cheng	2020	50%
Study summary Full title Isolation of extracellular vesicle with different precipitation-based methods exerts a tremendous impact on the biomarker analysis for clinical plasma samples All authors Cheng Peng, Jizhuang Wang, Qiyuan Bao, Jun Wang, Zhuochao Liu, Junxiang Wen, Weibin Zhang, Yuhui Shen Journal Cancer Biomarkers Abstract Background: Extracellular vesicles(EVs) is an emerging approach of cancer liquid biopsy. Although th (show more...)Background: Extracellular vesicles(EVs) is an emerging approach of cancer liquid biopsy. Although the precipitation-based method with commercial kits has gained popularity as the second most commonly used technique, these protocols vary tremendously with many included reagents still unknown to the community. Methods: In this study, we assigned each of the 3 clinical plasma samples into 6 aliquots to assess five commercial EV isolation kits, in comparison with ultracentrifugation(UC). We implemented a standardized EV preparation and transcriptome analysis workflow except the EV isolation methods used. The metrics of EVs and its RNA cargo (evRNA) were compared to assess the technical variations versus the biological variations in the clinical setting. Results: Although the size range of the isolated EVs demonstrated a similar distribution, we found significant technical variability among these methods, in terms of EV amount, purity, subpopulations and RNA integrity. Such variabilities were further relayed to a drastic divergence of evRNA expression on a transcriptome-wide fashion. Conclusions: Our study demonstrated a highly variable result from polymeric precipitation-based EV isolation methods, making EVs based biomarker analysis difficult to interpret and reproduce. We highlighted the importance of benchmarking and transparent reporting of the precipitation-based protocols in the liquid biopsy research. (hide) EV-METRIC 50% (83rd 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 Ribo Exosome Isolation Reagent Protein markers EV: TSG101/ CD81/ CD63/ CD9/ vimentin non-EV: calnexin/ Albumin/ Apolipoprotein A-1 Proteomics no Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Blood plasma Separation Method Commercial kit Other;Ribo Exosome Isolation Reagent Other Name other separation method Ribo Exosome Isolation Reagent 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 CD63/ TSG101/ CD81 Not detected EV-associated proteins vimentin/ CD9 Detected contaminants Apolipoprotein A-1/ Albumin Not detected contaminants calnexin Characterization: RNA analysis RNA analysis Type RNAsequencing Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 30-240 EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 86700000000

	EV200008	12/18	Homo sapiens	Blood plasma	Total Exosome Isolation	Peng, Cheng	2020	50%
Study summary Full title Isolation of extracellular vesicle with different precipitation-based methods exerts a tremendous impact on the biomarker analysis for clinical plasma samples All authors Cheng Peng, Jizhuang Wang, Qiyuan Bao, Jun Wang, Zhuochao Liu, Junxiang Wen, Weibin Zhang, Yuhui Shen Journal Cancer Biomarkers Abstract Background: Extracellular vesicles(EVs) is an emerging approach of cancer liquid biopsy. Although th (show more...)Background: Extracellular vesicles(EVs) is an emerging approach of cancer liquid biopsy. Although the precipitation-based method with commercial kits has gained popularity as the second most commonly used technique, these protocols vary tremendously with many included reagents still unknown to the community. Methods: In this study, we assigned each of the 3 clinical plasma samples into 6 aliquots to assess five commercial EV isolation kits, in comparison with ultracentrifugation(UC). We implemented a standardized EV preparation and transcriptome analysis workflow except the EV isolation methods used. The metrics of EVs and its RNA cargo (evRNA) were compared to assess the technical variations versus the biological variations in the clinical setting. Results: Although the size range of the isolated EVs demonstrated a similar distribution, we found significant technical variability among these methods, in terms of EV amount, purity, subpopulations and RNA integrity. Such variabilities were further relayed to a drastic divergence of evRNA expression on a transcriptome-wide fashion. Conclusions: Our study demonstrated a highly variable result from polymeric precipitation-based EV isolation methods, making EVs based biomarker analysis difficult to interpret and reproduce. We highlighted the importance of benchmarking and transparent reporting of the precipitation-based protocols in the liquid biopsy research. (hide) EV-METRIC 50% (83rd 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 Total Exosome Isolation Protein markers EV: TSG101/ CD81/ CD63/ CD9/ vimentin non-EV: calnexin/ Albumin/ Apolipoprotein A-1 Proteomics no Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Blood plasma Separation Method Commercial kit Total Exosome Isolation Other Name other separation method Total Exosome Isolation 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 CD63/ CD81 Not detected EV-associated proteins vimentin/ TSG101/ CD9 Detected contaminants Apolipoprotein A-1/ Albumin Not detected contaminants calnexin Characterization: RNA analysis RNA analysis Type RNAsequencing Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 30-240 EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 36700000000

	EV200008	13/18	Homo sapiens	Blood plasma	miRCURY Exosome Kit	Peng, Cheng	2020	50%
Study summary Full title Isolation of extracellular vesicle with different precipitation-based methods exerts a tremendous impact on the biomarker analysis for clinical plasma samples All authors Cheng Peng, Jizhuang Wang, Qiyuan Bao, Jun Wang, Zhuochao Liu, Junxiang Wen, Weibin Zhang, Yuhui Shen Journal Cancer Biomarkers Abstract Background: Extracellular vesicles(EVs) is an emerging approach of cancer liquid biopsy. Although th (show more...)Background: Extracellular vesicles(EVs) is an emerging approach of cancer liquid biopsy. Although the precipitation-based method with commercial kits has gained popularity as the second most commonly used technique, these protocols vary tremendously with many included reagents still unknown to the community. Methods: In this study, we assigned each of the 3 clinical plasma samples into 6 aliquots to assess five commercial EV isolation kits, in comparison with ultracentrifugation(UC). We implemented a standardized EV preparation and transcriptome analysis workflow except the EV isolation methods used. The metrics of EVs and its RNA cargo (evRNA) were compared to assess the technical variations versus the biological variations in the clinical setting. Results: Although the size range of the isolated EVs demonstrated a similar distribution, we found significant technical variability among these methods, in terms of EV amount, purity, subpopulations and RNA integrity. Such variabilities were further relayed to a drastic divergence of evRNA expression on a transcriptome-wide fashion. Conclusions: Our study demonstrated a highly variable result from polymeric precipitation-based EV isolation methods, making EVs based biomarker analysis difficult to interpret and reproduce. We highlighted the importance of benchmarking and transparent reporting of the precipitation-based protocols in the liquid biopsy research. (hide) EV-METRIC 50% (83rd 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 primary osteosarcoma 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 miRCURY Exosome Kit Protein markers EV: TSG101/ CD81/ CD63/ CD9/ vimentin non-EV: calnexin/ Albumin/ Apolipoprotein A-1 Proteomics no Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Blood plasma Separation Method Commercial kit Other;miRCURY Exosome Kit Other Name other separation method miRCURY Exosome Kit 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 CD63/ TSG101/ CD81 Not detected EV-associated proteins vimentin/ CD9 Detected contaminants Apolipoprotein A-1/ Albumin Not detected contaminants calnexin Characterization: RNA analysis RNA analysis Type RNAsequencing Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 30-240 EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 63300000000

	EV200008	14/18	Homo sapiens	Blood plasma	Ribo Exosome Isolation Reagent	Peng, Cheng	2020	50%
Study summary Full title Isolation of extracellular vesicle with different precipitation-based methods exerts a tremendous impact on the biomarker analysis for clinical plasma samples All authors Cheng Peng, Jizhuang Wang, Qiyuan Bao, Jun Wang, Zhuochao Liu, Junxiang Wen, Weibin Zhang, Yuhui Shen Journal Cancer Biomarkers Abstract Background: Extracellular vesicles(EVs) is an emerging approach of cancer liquid biopsy. Although th (show more...)Background: Extracellular vesicles(EVs) is an emerging approach of cancer liquid biopsy. Although the precipitation-based method with commercial kits has gained popularity as the second most commonly used technique, these protocols vary tremendously with many included reagents still unknown to the community. Methods: In this study, we assigned each of the 3 clinical plasma samples into 6 aliquots to assess five commercial EV isolation kits, in comparison with ultracentrifugation(UC). We implemented a standardized EV preparation and transcriptome analysis workflow except the EV isolation methods used. The metrics of EVs and its RNA cargo (evRNA) were compared to assess the technical variations versus the biological variations in the clinical setting. Results: Although the size range of the isolated EVs demonstrated a similar distribution, we found significant technical variability among these methods, in terms of EV amount, purity, subpopulations and RNA integrity. Such variabilities were further relayed to a drastic divergence of evRNA expression on a transcriptome-wide fashion. Conclusions: Our study demonstrated a highly variable result from polymeric precipitation-based EV isolation methods, making EVs based biomarker analysis difficult to interpret and reproduce. We highlighted the importance of benchmarking and transparent reporting of the precipitation-based protocols in the liquid biopsy research. (hide) EV-METRIC 50% (83rd 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 primary osteosarcoma 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 Ribo Exosome Isolation Reagent Protein markers EV: TSG101/ CD81/ CD63/ CD9/ vimentin non-EV: calnexin/ Albumin/ Apolipoprotein A-1 Proteomics no Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Blood plasma Separation Method Commercial kit Other;Ribo Exosome Isolation Reagent Other Name other separation method Ribo Exosome Isolation Reagent 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 vimentin/ CD63/ TSG101/ CD81 Not detected EV-associated proteins CD9 Detected contaminants Apolipoprotein A-1/ Albumin Not detected contaminants calnexin Characterization: RNA analysis RNA analysis Type RNAsequencing Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 30-240 EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 70000000000

	EV200008	15/18	Homo sapiens	Blood plasma	Total Exosome Isolation	Peng, Cheng	2020	50%
Study summary Full title Isolation of extracellular vesicle with different precipitation-based methods exerts a tremendous impact on the biomarker analysis for clinical plasma samples All authors Cheng Peng, Jizhuang Wang, Qiyuan Bao, Jun Wang, Zhuochao Liu, Junxiang Wen, Weibin Zhang, Yuhui Shen Journal Cancer Biomarkers Abstract Background: Extracellular vesicles(EVs) is an emerging approach of cancer liquid biopsy. Although th (show more...)Background: Extracellular vesicles(EVs) is an emerging approach of cancer liquid biopsy. Although the precipitation-based method with commercial kits has gained popularity as the second most commonly used technique, these protocols vary tremendously with many included reagents still unknown to the community. Methods: In this study, we assigned each of the 3 clinical plasma samples into 6 aliquots to assess five commercial EV isolation kits, in comparison with ultracentrifugation(UC). We implemented a standardized EV preparation and transcriptome analysis workflow except the EV isolation methods used. The metrics of EVs and its RNA cargo (evRNA) were compared to assess the technical variations versus the biological variations in the clinical setting. Results: Although the size range of the isolated EVs demonstrated a similar distribution, we found significant technical variability among these methods, in terms of EV amount, purity, subpopulations and RNA integrity. Such variabilities were further relayed to a drastic divergence of evRNA expression on a transcriptome-wide fashion. Conclusions: Our study demonstrated a highly variable result from polymeric precipitation-based EV isolation methods, making EVs based biomarker analysis difficult to interpret and reproduce. We highlighted the importance of benchmarking and transparent reporting of the precipitation-based protocols in the liquid biopsy research. (hide) EV-METRIC 50% (83rd 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 primary osteosarcoma 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 Total Exosome Isolation Protein markers EV: TSG101/ CD81/ CD63/ vimentin non-EV: calnexin/ Albumin/ Apolipoprotein A-1 Proteomics no Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Blood plasma Separation Method Commercial kit Total Exosome Isolation Other Name other separation method Total Exosome Isolation 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 vimentin/ CD63/ CD81 Not detected EV-associated proteins TSG101/ CD63 Detected contaminants Apolipoprotein A-1/ Albumin Not detected contaminants calnexin Characterization: RNA analysis RNA analysis Type RNAsequencing Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 30-240 EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 103000000000

	EV200008	16/18	Homo sapiens	Blood plasma	miRCURY Exosome Kit	Peng, Cheng	2020	50%
Study summary Full title Isolation of extracellular vesicle with different precipitation-based methods exerts a tremendous impact on the biomarker analysis for clinical plasma samples All authors Cheng Peng, Jizhuang Wang, Qiyuan Bao, Jun Wang, Zhuochao Liu, Junxiang Wen, Weibin Zhang, Yuhui Shen Journal Cancer Biomarkers Abstract Background: Extracellular vesicles(EVs) is an emerging approach of cancer liquid biopsy. Although th (show more...)Background: Extracellular vesicles(EVs) is an emerging approach of cancer liquid biopsy. Although the precipitation-based method with commercial kits has gained popularity as the second most commonly used technique, these protocols vary tremendously with many included reagents still unknown to the community. Methods: In this study, we assigned each of the 3 clinical plasma samples into 6 aliquots to assess five commercial EV isolation kits, in comparison with ultracentrifugation(UC). We implemented a standardized EV preparation and transcriptome analysis workflow except the EV isolation methods used. The metrics of EVs and its RNA cargo (evRNA) were compared to assess the technical variations versus the biological variations in the clinical setting. Results: Although the size range of the isolated EVs demonstrated a similar distribution, we found significant technical variability among these methods, in terms of EV amount, purity, subpopulations and RNA integrity. Such variabilities were further relayed to a drastic divergence of evRNA expression on a transcriptome-wide fashion. Conclusions: Our study demonstrated a highly variable result from polymeric precipitation-based EV isolation methods, making EVs based biomarker analysis difficult to interpret and reproduce. We highlighted the importance of benchmarking and transparent reporting of the precipitation-based protocols in the liquid biopsy research. (hide) EV-METRIC 50% (83rd 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 Metastatic osteosarcoma 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 miRCURY Exosome Kit Protein markers EV: TSG101/ CD81/ CD63/ CD9/ vimentin non-EV: calnexin/ Albumin/ Apolipoprotein A-1 Proteomics no Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Blood plasma Separation Method Commercial kit Other;miRCURY Exosome Kit Other Name other separation method miRCURY Exosome Kit 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 vimentin/ TSG101/ CD63/ CD81 Not detected EV-associated proteins CD9 Detected contaminants Apolipoprotein A-1/ Albumin Not detected contaminants calnexin Characterization: RNA analysis RNA analysis Type RNAsequencing Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 30-240 EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 60000000000

	EV200008	17/18	Homo sapiens	Blood plasma	Ribo Exosome Isolation Reagent	Peng, Cheng	2020	50%
Study summary Full title Isolation of extracellular vesicle with different precipitation-based methods exerts a tremendous impact on the biomarker analysis for clinical plasma samples All authors Cheng Peng, Jizhuang Wang, Qiyuan Bao, Jun Wang, Zhuochao Liu, Junxiang Wen, Weibin Zhang, Yuhui Shen Journal Cancer Biomarkers Abstract Background: Extracellular vesicles(EVs) is an emerging approach of cancer liquid biopsy. Although th (show more...)Background: Extracellular vesicles(EVs) is an emerging approach of cancer liquid biopsy. Although the precipitation-based method with commercial kits has gained popularity as the second most commonly used technique, these protocols vary tremendously with many included reagents still unknown to the community. Methods: In this study, we assigned each of the 3 clinical plasma samples into 6 aliquots to assess five commercial EV isolation kits, in comparison with ultracentrifugation(UC). We implemented a standardized EV preparation and transcriptome analysis workflow except the EV isolation methods used. The metrics of EVs and its RNA cargo (evRNA) were compared to assess the technical variations versus the biological variations in the clinical setting. Results: Although the size range of the isolated EVs demonstrated a similar distribution, we found significant technical variability among these methods, in terms of EV amount, purity, subpopulations and RNA integrity. Such variabilities were further relayed to a drastic divergence of evRNA expression on a transcriptome-wide fashion. Conclusions: Our study demonstrated a highly variable result from polymeric precipitation-based EV isolation methods, making EVs based biomarker analysis difficult to interpret and reproduce. We highlighted the importance of benchmarking and transparent reporting of the precipitation-based protocols in the liquid biopsy research. (hide) EV-METRIC 50% (83rd 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 Metastatic osteosarcoma 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 Ribo Exosome Isolation Reagent Protein markers EV: TSG101/ CD81/ CD63/ CD9/ vimentin non-EV: calnexin/ Albumin/ Apolipoprotein A-1 Proteomics no Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Blood plasma Separation Method Commercial kit Other;Ribo Exosome Isolation Reagent Other Name other separation method Ribo Exosome Isolation Reagent 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 vimentin/ CD63/ TSG101/ CD81 Not detected EV-associated proteins CD9 Detected contaminants Apolipoprotein A-1/ Albumin Not detected contaminants calnexin Characterization: RNA analysis RNA analysis Type RNAsequencing Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 30-240 EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 160000000000

	EV200008	18/18	Homo sapiens	Blood plasma	Total Exosome Isolation	Peng, Cheng	2020	50%
Study summary Full title Isolation of extracellular vesicle with different precipitation-based methods exerts a tremendous impact on the biomarker analysis for clinical plasma samples All authors Cheng Peng, Jizhuang Wang, Qiyuan Bao, Jun Wang, Zhuochao Liu, Junxiang Wen, Weibin Zhang, Yuhui Shen Journal Cancer Biomarkers Abstract Background: Extracellular vesicles(EVs) is an emerging approach of cancer liquid biopsy. Although th (show more...)Background: Extracellular vesicles(EVs) is an emerging approach of cancer liquid biopsy. Although the precipitation-based method with commercial kits has gained popularity as the second most commonly used technique, these protocols vary tremendously with many included reagents still unknown to the community. Methods: In this study, we assigned each of the 3 clinical plasma samples into 6 aliquots to assess five commercial EV isolation kits, in comparison with ultracentrifugation(UC). We implemented a standardized EV preparation and transcriptome analysis workflow except the EV isolation methods used. The metrics of EVs and its RNA cargo (evRNA) were compared to assess the technical variations versus the biological variations in the clinical setting. Results: Although the size range of the isolated EVs demonstrated a similar distribution, we found significant technical variability among these methods, in terms of EV amount, purity, subpopulations and RNA integrity. Such variabilities were further relayed to a drastic divergence of evRNA expression on a transcriptome-wide fashion. Conclusions: Our study demonstrated a highly variable result from polymeric precipitation-based EV isolation methods, making EVs based biomarker analysis difficult to interpret and reproduce. We highlighted the importance of benchmarking and transparent reporting of the precipitation-based protocols in the liquid biopsy research. (hide) EV-METRIC 50% (83rd 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 Metastatic osteosarcoma 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 Total Exosome Isolation Protein markers EV: TSG101/ CD81/ CD63/ CD9/ vimentin non-EV: calnexin Proteomics no Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Blood plasma Separation Method Commercial kit Total Exosome Isolation Other Name other separation method Total Exosome Isolation 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 vimentin/ TSG101/ CD81 Not detected EV-associated proteins CD63/ CD9 Not detected contaminants calnexin Characterization: RNA analysis RNA analysis Type RNAsequencing Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 30-240 EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 107000000000

	EV190100	1/10	Homo sapiens	CNE1	ExoQuick	Chaoliang Liao	2020	50%
Study summary Full title Epstein-Barr virus-encoded latent membrane protein 1 promotes extracellular vesicle secretion through syndecan-2 and synaptotagmin-like-4 in nasopharyngeal carcinoma cells All authors Chaoliang Liao, Qin Zhou, Zhibao Zhang, Xia Wu, Zhuan Zhou, Bo Li, Jinwu Peng, Liangfang Shen, Dan Li, Xiangjian Luo, Lifang Yang Journal J Pharm Sci Abstract Increasing evidence indicates that extracellular vesicles (EVs) play an important role in cancer cel (show more...)Increasing evidence indicates that extracellular vesicles (EVs) play an important role in cancer cell-to-cell communication. The Epstein-Barr virus (EBV)-encoded latent membrane protein 1 (LMP1), which is closely associated with nasopharyngeal carcinoma (NPC) pathogenesis, can trigger multiple cell signaling pathways that affect cell progression. Several reports have shown that LMP1 promotes EV secretion, and LMP1 trafficking by EVs can enhances cancer progression and metastasis. However, the molecular mechanism by which LMP1 promotes EV secretion is not well understood. In the present study, we found that LMP1 promotes EV secretion by upregulated syndecan-2 (SDC2) and synaptotagmin-like-4 (SYTL4) through nuclear factor (NF)-κB signaling in NPC cells. Further study indicated that SDC2 interacted with syntenin, which promoted the formation of the EVs, and SYTL4 is associated with the release of EVs. Moreover, we found that stimulation of EV secretion by LMP1 can enhance the proliferation and invasion ability of recipient NPC cells and tumor growth in vivo. In summary, we found a new mechanism by which LMP1 upregulates SDC2 and SYTL4 through NF-κB signaling to promote EV secretion, and further enhance cancer progression of NPC. (hide) EV-METRIC 50% (87th 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 ExoQuick Protein markers EV: HSP70/ CD63 non-EV: Calnexin Proteomics no Show all info Study aim Function/Biogenesis/cargo sorting/Mechanism of uptake/transfer Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells CNE1 EV-harvesting Medium Serum-containing, but physical separation of serum EVs and secreted EVs (e.g. Bioreactor flask) Separation Method Commercial kit ExoQuick Other Name other separation method ExoQuick 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 CD63/ HSP70 Not detected contaminants Calnexin Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 21.04-255.6 EV concentration Yes EM EM-type Transmission-EM Image type Close-up Report size (nm) 80-100

	EV190100	2/10	Homo sapiens	CNE1-LMP1	ExoQuick	Chaoliang Liao	2020	50%
Study summary Full title Epstein-Barr virus-encoded latent membrane protein 1 promotes extracellular vesicle secretion through syndecan-2 and synaptotagmin-like-4 in nasopharyngeal carcinoma cells All authors Chaoliang Liao, Qin Zhou, Zhibao Zhang, Xia Wu, Zhuan Zhou, Bo Li, Jinwu Peng, Liangfang Shen, Dan Li, Xiangjian Luo, Lifang Yang Journal J Pharm Sci Abstract Increasing evidence indicates that extracellular vesicles (EVs) play an important role in cancer cel (show more...)Increasing evidence indicates that extracellular vesicles (EVs) play an important role in cancer cell-to-cell communication. The Epstein-Barr virus (EBV)-encoded latent membrane protein 1 (LMP1), which is closely associated with nasopharyngeal carcinoma (NPC) pathogenesis, can trigger multiple cell signaling pathways that affect cell progression. Several reports have shown that LMP1 promotes EV secretion, and LMP1 trafficking by EVs can enhances cancer progression and metastasis. However, the molecular mechanism by which LMP1 promotes EV secretion is not well understood. In the present study, we found that LMP1 promotes EV secretion by upregulated syndecan-2 (SDC2) and synaptotagmin-like-4 (SYTL4) through nuclear factor (NF)-κB signaling in NPC cells. Further study indicated that SDC2 interacted with syntenin, which promoted the formation of the EVs, and SYTL4 is associated with the release of EVs. Moreover, we found that stimulation of EV secretion by LMP1 can enhance the proliferation and invasion ability of recipient NPC cells and tumor growth in vivo. In summary, we found a new mechanism by which LMP1 upregulates SDC2 and SYTL4 through NF-κB signaling to promote EV secretion, and further enhance cancer progression of NPC. (hide) EV-METRIC 50% (87th 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 ExoQuick Protein markers EV: HSP70/ CD63 non-EV: Calnexin Proteomics no Show all info Study aim Function/Biogenesis/cargo sorting/Mechanism of uptake/transfer Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells CNE1-LMP1 EV-harvesting Medium Serum-containing, but physical separation of serum EVs and secreted EVs (e.g. Bioreactor flask) Separation Method Commercial kit ExoQuick Other Name other separation method ExoQuick 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 CD63/ HSP70 Not detected contaminants Calnexin Characterization: Lipid analysis No EM EM-type Transmission-EM Image type Close-up Report size (nm) 80-100

	EV190100	6/10	Homo sapiens	HKI	ExoQuick	Chaoliang Liao	2020	50%
Study summary Full title Epstein-Barr virus-encoded latent membrane protein 1 promotes extracellular vesicle secretion through syndecan-2 and synaptotagmin-like-4 in nasopharyngeal carcinoma cells All authors Chaoliang Liao, Qin Zhou, Zhibao Zhang, Xia Wu, Zhuan Zhou, Bo Li, Jinwu Peng, Liangfang Shen, Dan Li, Xiangjian Luo, Lifang Yang Journal J Pharm Sci Abstract Increasing evidence indicates that extracellular vesicles (EVs) play an important role in cancer cel (show more...)Increasing evidence indicates that extracellular vesicles (EVs) play an important role in cancer cell-to-cell communication. The Epstein-Barr virus (EBV)-encoded latent membrane protein 1 (LMP1), which is closely associated with nasopharyngeal carcinoma (NPC) pathogenesis, can trigger multiple cell signaling pathways that affect cell progression. Several reports have shown that LMP1 promotes EV secretion, and LMP1 trafficking by EVs can enhances cancer progression and metastasis. However, the molecular mechanism by which LMP1 promotes EV secretion is not well understood. In the present study, we found that LMP1 promotes EV secretion by upregulated syndecan-2 (SDC2) and synaptotagmin-like-4 (SYTL4) through nuclear factor (NF)-κB signaling in NPC cells. Further study indicated that SDC2 interacted with syntenin, which promoted the formation of the EVs, and SYTL4 is associated with the release of EVs. Moreover, we found that stimulation of EV secretion by LMP1 can enhance the proliferation and invasion ability of recipient NPC cells and tumor growth in vivo. In summary, we found a new mechanism by which LMP1 upregulates SDC2 and SYTL4 through NF-κB signaling to promote EV secretion, and further enhance cancer progression of NPC. (hide) EV-METRIC 50% (87th 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 ExoQuick Protein markers EV: HSP70/ CD63 non-EV: Calnexin Proteomics no Show all info Study aim Function/Biogenesis/cargo sorting/Mechanism of uptake/transfer Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells HKI EV-harvesting Medium Serum-containing, but physical separation of serum EVs and secreted EVs (e.g. Bioreactor flask) Separation Method Commercial kit ExoQuick Other Name other separation method ExoQuick 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 CD63/ HSP70 Not detected contaminants Calnexin Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 24.3-199.2 EV concentration Yes EM EM-type Transmission-EM Image type Close-up Report size (nm) 80-100

	EV190100	8/10	Homo sapiens	C666-1	ExoQuick	Chaoliang Liao	2020	50%
Study summary Full title Epstein-Barr virus-encoded latent membrane protein 1 promotes extracellular vesicle secretion through syndecan-2 and synaptotagmin-like-4 in nasopharyngeal carcinoma cells All authors Chaoliang Liao, Qin Zhou, Zhibao Zhang, Xia Wu, Zhuan Zhou, Bo Li, Jinwu Peng, Liangfang Shen, Dan Li, Xiangjian Luo, Lifang Yang Journal J Pharm Sci Abstract Increasing evidence indicates that extracellular vesicles (EVs) play an important role in cancer cel (show more...)Increasing evidence indicates that extracellular vesicles (EVs) play an important role in cancer cell-to-cell communication. The Epstein-Barr virus (EBV)-encoded latent membrane protein 1 (LMP1), which is closely associated with nasopharyngeal carcinoma (NPC) pathogenesis, can trigger multiple cell signaling pathways that affect cell progression. Several reports have shown that LMP1 promotes EV secretion, and LMP1 trafficking by EVs can enhances cancer progression and metastasis. However, the molecular mechanism by which LMP1 promotes EV secretion is not well understood. In the present study, we found that LMP1 promotes EV secretion by upregulated syndecan-2 (SDC2) and synaptotagmin-like-4 (SYTL4) through nuclear factor (NF)-κB signaling in NPC cells. Further study indicated that SDC2 interacted with syntenin, which promoted the formation of the EVs, and SYTL4 is associated with the release of EVs. Moreover, we found that stimulation of EV secretion by LMP1 can enhance the proliferation and invasion ability of recipient NPC cells and tumor growth in vivo. In summary, we found a new mechanism by which LMP1 upregulates SDC2 and SYTL4 through NF-κB signaling to promote EV secretion, and further enhance cancer progression of NPC. (hide) EV-METRIC 50% (87th 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 ExoQuick Protein markers EV: HSP70/ CD63 non-EV: Calnexin Proteomics no Show all info Study aim Function/Biogenesis/cargo sorting/Mechanism of uptake/transfer Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells C666-1 EV-harvesting Medium Serum-containing, but physical separation of serum EVs and secreted EVs (e.g. Bioreactor flask) Separation Method Commercial kit ExoQuick Other Name other separation method ExoQuick 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 CD63/ HSP70 Not detected contaminants Calnexin Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 13.54-225.9 EV concentration Yes EM EM-type Transmission-EM Image type Close-up Report size (nm) 80-100

	EV190099	1/3	Mus musculus	Serum	Filtration qEV	Anastasi, Federica	2020	50%
Study summary Full title Proteomics analysis of serum small extracellular vesicles for the longitudinal study of a glioblastoma multiforme mouse model All authors Federica Anastasi, Francesco Greco, Marialaura Dilillo, Eleonora Vannini, Valentina Cappello, Laura Baroncelli, Mario Costa, Mauro Gemmi, Matteo Caleo & Liam A. McDonnell Journal Sci Rep Abstract Longitudinal analysis of disease models enables the molecular changes due to disease progression or (show more...)Longitudinal analysis of disease models enables the molecular changes due to disease progression or therapeutic intervention to be better resolved. Approximately 75 µl of serum can be drawn from a mouse every 14 days. To date no methods have been reported that are able to analyze the proteome of small extracellular vesicles (sEV’s) from such low serum volumes. Here we report a method for the proteomics analysis of sEV's from 50 µl of serum. Two sEV isolation procedures were first compared; precipitation based purification (PPT) and size exclusion chromatography (SEC). The methodological comparison confirmed that SEC led to purer sEV’s both in terms of size and identified proteins. The procedure was then scaled down and the proteolytic digestion further optimized. The method was then applied to a longitudinal study of serum-sEV proteome changes in a glioblastoma multiforme (GBM) mouse model. Serum was collected at multiple time points, sEV’s isolated and their proteins analyzed. The protocol enabled 274 protein groups to be identified and quantified. The longitudinal analysis revealed 25 deregulated proteins in GBM serum sEV's including proteins previously shown to be associated with GBM progression and metastasis (Myh9, Tln-1, Angpt1, Thbs1). (hide) EV-METRIC 50% (89th 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 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 Filtration qEV Protein markers EV: non-EV: Proteomics yes Show all info Study aim New methodological development/Biomarker/Identification of content (omics approaches) Sample Species Mus musculus Sample Type Serum Separation Method Filtration steps 0.22µm or 0.2µm Commercial kit qEV Other Name other separation method qEV Characterization: Protein analysis Protein Concentration Method microBCA Proteomics database No Characterization: Lipid analysis No EM EM-type Transmission-EM Image type Close-up, Wide-field Report size (nm) 45

	EV190099	2/3	Mus musculus	Serum	Filtration Total Exosome Isolation	Anastasi, Federica	2020	50%
Study summary Full title Proteomics analysis of serum small extracellular vesicles for the longitudinal study of a glioblastoma multiforme mouse model All authors Federica Anastasi, Francesco Greco, Marialaura Dilillo, Eleonora Vannini, Valentina Cappello, Laura Baroncelli, Mario Costa, Mauro Gemmi, Matteo Caleo & Liam A. McDonnell Journal Sci Rep Abstract Longitudinal analysis of disease models enables the molecular changes due to disease progression or (show more...)Longitudinal analysis of disease models enables the molecular changes due to disease progression or therapeutic intervention to be better resolved. Approximately 75 µl of serum can be drawn from a mouse every 14 days. To date no methods have been reported that are able to analyze the proteome of small extracellular vesicles (sEV’s) from such low serum volumes. Here we report a method for the proteomics analysis of sEV's from 50 µl of serum. Two sEV isolation procedures were first compared; precipitation based purification (PPT) and size exclusion chromatography (SEC). The methodological comparison confirmed that SEC led to purer sEV’s both in terms of size and identified proteins. The procedure was then scaled down and the proteolytic digestion further optimized. The method was then applied to a longitudinal study of serum-sEV proteome changes in a glioblastoma multiforme (GBM) mouse model. Serum was collected at multiple time points, sEV’s isolated and their proteins analyzed. The protocol enabled 274 protein groups to be identified and quantified. The longitudinal analysis revealed 25 deregulated proteins in GBM serum sEV's including proteins previously shown to be associated with GBM progression and metastasis (Myh9, Tln-1, Angpt1, Thbs1). (hide) EV-METRIC 50% (89th 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 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 Filtration Total Exosome Isolation Protein markers EV: non-EV: Proteomics yes Show all info Study aim New methodological development/Biomarker/Identification of content (omics approaches) Sample Species Mus musculus Sample Type Serum Separation Method Filtration steps 0.22µm or 0.2µm Commercial kit Total Exosome Isolation Other Name other separation method Total Exosome Isolation Characterization: Protein analysis Protein Concentration Method microBCA Proteomics database No Characterization: Lipid analysis No EM EM-type Transmission-EM Image type Close-up, Wide-field Report size (nm) 37

	EV190091	1/1	Homo sapiens	JAR ATCC HTB-144	(d)(U)C SEC	Getnet Midekessa	2020	50%
Study summary Full title Zeta Potential of Extracellular Vesicles: Toward Understanding the Attributes that Determine Colloidal Stability All authors Getnet Midekessa, Kasun Godakumara, James Ord, Janeli Viil, Freddy Lättekivi, Keerthie Dissanayake, Sergei Kopanchuk, Ago Rinken, Aneta Andronowska, Sourav Bhattacharjee, Toonika Rinken, Alireza Fazeli Journal ACS Omega Abstract Extracellular vesicles (EVs), including exosomes and microvesicles (<200 nm), play a vital role in i (show more...)Extracellular vesicles (EVs), including exosomes and microvesicles (<200 nm), play a vital role in intercellular communication and carry a net negative surface charge under physiological conditions. Zeta potential (ZP) is a popular method to measure the surface potential of EVs, while used as an indicator of surface charge, and colloidal stability influenced by surface chemistry, bioconjugation, and the theoretical model applied. Here, we investigated the effects of such factors on ZP of well-characterized EVs derived from the human choriocarcinoma JAr cells. The EVs were suspended in phosphate-buffered saline (PBS) of various phosphate ionic concentrations (0.01, 0.1, and 1 mM), with or without detergent (Tween-20), or in the presence (10 mM) of different salts (NaCl, KCl, CaCl2, and AlCl3) and at different pH values (4, 7, and 10) while the ZP was measured. The ZP changed inversely with the buffer concentration, while Tween-20 caused a significant (p < 0.05) lowering of the ZP. Moreover, the ZP was significantly (p < 0.05) less negative in the presence of ions with higher valency (Al3+/Ca2+) than in the presence of monovalent ones (Na+/K+). Besides, the ZP of EVs became less negative at acidic pH, and vice versa. The integrated data underpins the crucial role of physicochemical attributes that influence the colloidal stability of EVs. (hide) EV-METRIC 50% (87th 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 SEC Protein markers EV: CD81/ HSP70/ CD63/ CD9 non-EV: Proteomics no 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 JAR ATCC HTB-144 EV-harvesting Medium EV-depleted medium Preparation of EDS Not specified Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Pelleting performed No Size-exclusion chromatography Total column volume (mL) 10.5 Sample volume/column (mL) 0.5 Resin type Sepharose 4B 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/ HSP70/ CD81 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 15 - 500 EV concentration Yes EM EM-type Transmission-EM/ Scanning-EM Image type Wide-field Report size (nm) 120 -200

	EV190075	1/3	Homo sapiens	mesenchymal stem cells	Filtration Total Exosome Isolation UF	Thomas E. Whittaker	2020	50%
Study summary Full title Experimental artefacts can lead to misattribution of bioactivity from soluble mesenchymal stem cell paracrine factors to extracellular vesicles All authors Thomas E. Whittaker, Anika Nagelkerke , Valeria Nele , Ulrike Kauscher & Molly M. Stevens Journal J Extracell Vesicles Abstract It has been demonstrated that some commonly used Extracellular Vesicle (EV) isolation techniques can (show more...)It has been demonstrated that some commonly used Extracellular Vesicle (EV) isolation techniques can lead to substantial contamination with non-EV factors. Whilst it has been established that this impacts the identification of biomarkers, the impact on apparent EV bioactivity has not been explored. Extracellular vesicles have been implicated as critical mediators of therapeutic human mesenchymal stem cell (hMSC) paracrine signalling. Isolated hMSC-EVs have been used to treat multiple in vitro and in vivo models of tissue damage. However, the relative contributions of EVs and non-EV factors have not been directly compared. The dependence of hMSC paracrine signalling on EVs was first established by ultrafiltration of hMSC-conditioned medium to deplete EVs, which led to a loss of signalling activity. Here, we show that this method also causes depletion of non-EV factors, and that when this is prevented proangiogenic signalling activity is fully restored in vitro. Subsequently, we used size-exclusion chromatography (SEC) to separate EVs and soluble proteins to directly and quantitatively compare their relative contributions to signalling. Non-EV factors were found to be necessary and sufficient for the stimulation of angiogenesis and wound healing in vitro. EVs in isolation were found to be capable of potentiating signalling only when isolated by a low-purity method, or when used at comparatively high concentrations. These results indicate a potential for contaminating soluble factors to artefactually increase the apparent bioactivity of EV isolates and could have implications for future studies on the biological roles of EVs. (hide) EV-METRIC 50% (87th 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 Filtration Total Exosome Isolation UF Protein markers EV: / CD81/ CD63/ CD9 non-EV: VEGF Proteomics no Show all info Study aim Function/New methodological development/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells mesenchymal stem cells EV-harvesting Medium Serum free medium Separation Method Filtration steps 0.45µm > x > 0.22µm, Ultra filtration Cut-off size (kDa) 3 Membrane type Regenerated cellulose Commercial kit Total Exosome Isolation Other Name other separation method Total Exosome Isolation Characterization: Protein analysis Protein Concentration Method Fluorometric assay (e.g. Qubit, NanoOrange,...) ELISA Antibody details provided? No Detected EV-associated proteins Not detected EV-associated proteins Detected contaminants VEGF Not detected contaminants CD63/ CD81/ CD9 Other 1 Dot Blot Detected EV-associated proteins CD63/ CD81/ CD9 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Modus Reported size (nm) 115.8 EV concentration Yes

	EV190075	2/3	Homo sapiens	mesenchymal stem cells	Filtration SEC SEC (non-commercial) UF	Thomas E. Whittaker	2020	50%
Study summary Full title Experimental artefacts can lead to misattribution of bioactivity from soluble mesenchymal stem cell paracrine factors to extracellular vesicles All authors Thomas E. Whittaker, Anika Nagelkerke , Valeria Nele , Ulrike Kauscher & Molly M. Stevens Journal J Extracell Vesicles Abstract It has been demonstrated that some commonly used Extracellular Vesicle (EV) isolation techniques can (show more...)It has been demonstrated that some commonly used Extracellular Vesicle (EV) isolation techniques can lead to substantial contamination with non-EV factors. Whilst it has been established that this impacts the identification of biomarkers, the impact on apparent EV bioactivity has not been explored. Extracellular vesicles have been implicated as critical mediators of therapeutic human mesenchymal stem cell (hMSC) paracrine signalling. Isolated hMSC-EVs have been used to treat multiple in vitro and in vivo models of tissue damage. However, the relative contributions of EVs and non-EV factors have not been directly compared. The dependence of hMSC paracrine signalling on EVs was first established by ultrafiltration of hMSC-conditioned medium to deplete EVs, which led to a loss of signalling activity. Here, we show that this method also causes depletion of non-EV factors, and that when this is prevented proangiogenic signalling activity is fully restored in vitro. Subsequently, we used size-exclusion chromatography (SEC) to separate EVs and soluble proteins to directly and quantitatively compare their relative contributions to signalling. Non-EV factors were found to be necessary and sufficient for the stimulation of angiogenesis and wound healing in vitro. EVs in isolation were found to be capable of potentiating signalling only when isolated by a low-purity method, or when used at comparatively high concentrations. These results indicate a potential for contaminating soluble factors to artefactually increase the apparent bioactivity of EV isolates and could have implications for future studies on the biological roles of EVs. (hide) EV-METRIC 50% (87th 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 Filtration SEC Size-exclusion chromatography (non-commercial) UF Protein markers EV: / CD81/ CD63/ CD9 non-EV: VEGF Proteomics no Show all info Study aim Function/New methodological development/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells mesenchymal stem cells EV-harvesting Medium Serum free medium Separation Method Filtration steps 0.45µm > x > 0.22µm, Ultra filtration Cut-off size (kDa) 3 Membrane type Regenerated cellulose Size-exclusion chromatography Total column volume (mL) 22 Sample volume/column (mL) 0.5 Resin type Sepharose CL-2B Other Name other separation method Characterization: Protein analysis Protein Concentration Method Fluorometric assay (e.g. Qubit, NanoOrange,...) ELISA Antibody details provided? No Detected EV-associated proteins Not detected EV-associated proteins Not detected contaminants CD63/ CD81/ CD9 Other 1 Dot Blot Detected EV-associated proteins CD63/ CD81/ CD9 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Modus Reported size (nm) 125 EV concentration Yes

	EV190064	4/10	Homo sapiens	Urine	(d)(U)C ExoQuick UF	Dhondt B	2020	50%
Study summary Full title Unravelling the proteomic landscape of extracellular vesicles in prostate cancer by density-based fractionation of urine. All authors Dhondt B, Geeurickx E, Tulkens J, Van Deun J, Vergauwen G, Lippens L, Miinalainen I, Rappu P, Heino J, Ost P, Lumen N, De Wever O, Hendrix A. Journal J Extracell Vesicles Abstract Extracellular vesicles (EV) are increasingly being recognized as important vehicles of intercellular (show more...)Extracellular vesicles (EV) are increasingly being recognized as important vehicles of intercellular communication and promising diagnostic and prognostic biomarkers in cancer. Despite this enormous clinical potential, the plethora of methods to separate EV from biofluids, providing material of highly variable purity, and lacking knowledge regarding methodological repeatability pose a barrier to clinical translation. Urine is considered an ideal proximal fluid for the study of EV in urological cancers due to its direct contact with the urogenital system. We demonstrate that density-based fractionation of urine by bottom-up Optiprep density gradient centrifugation separates EV and soluble proteins with high specificity and repeatability. Mass spectrometry-based proteomic analysis of urinary EV (uEV) in men with benign and malignant prostate disease allowed us to significantly expand the known human uEV proteome with high specificity and identifies a unique biological profile in prostate cancer not uncovered by the analysis of soluble proteins. In addition, profiling the proteome of EV separated from prostate tumour conditioned medium and matched uEV confirms the specificity of the identified uEV proteome for prostate cancer. Finally, a comparative proteomic analysis with uEV from patients with bladder and renal cancer provided additional evidence of the selective enrichment of protein signatures in uEV reflecting their respective cancer tissues of origin. In conclusion, this study identifies hundreds of previously undetected proteins in uEV of prostate cancer patients and provides a powerful toolbox to map uEV content and contaminants ultimately allowing biomarker discovery in urological cancers. (hide) EV-METRIC 50% (86th 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 ExoQuick UF Protein markers EV: Alix/ Flotillin1/ CD9 non-EV: Tamm-Horsfall protein Proteomics no Show all info Study aim Function/New methodological development/Biomarker/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 Between 800 g and 10,000 g Pelleting performed No Ultra filtration Cut-off size (kDa) 10 Membrane type Regenerated cellulose Commercial kit ExoQuick Other Name other separation method ExoQuick Characterization: Protein analysis Protein Concentration Method Fluorometric assay (e.g. Qubit, NanoOrange,...) Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins Alix/ CD9 Not detected EV-associated proteins Flotillin1 Detected contaminants Tamm-Horsfall protein Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 185.4 EV concentration Yes

	EV210069	3/4	Homo sapiens	Blood plasma	SEC (non-commercial) UF (d)(U)C	Levine, Lisa	2020	45%
Study summary Full title Syncytiotrophoblast extracellular microvesicle profiles in maternal circulation for noninvasive diagnosis of preeclampsia All authors Lisa Levine, Andreas Habertheuer, Chirag Ram, Laxminarayana Korutla, Nadav Schwartz, Robert W Hu, Sanjana Reddy, Andrew Freas, Patrick D Zielinski, Joey Harmon, Sudheer Kumar Molugu, Samuel Parry, Prashanth Vallabhajosyula Journal Sci Rep Abstract Preeclampsia is the most common placental pathology in pregnant females, with increased morbidity an (show more...)Preeclampsia is the most common placental pathology in pregnant females, with increased morbidity and mortality incurred on the mother and the fetus. There is a need for improved biomarkers for diagnosis and monitoring of this condition. Placental syncytiotrophoblasts at the maternal-fetal interface release nanoparticles, including extracellular microvesicles, into the maternal blood during pregnancy. Syncytiotrophoblast extracellular microvesicles (STEVs) are being studied for their diagnostic potential and for their potential physiologic role in preeclampsia. We hypothesized that STEV profiles in maternal circulation would be altered under conditions of preeclampsia compared to normal pregnancy. Extracellular vesicles (EVs) released by BeWo cells in vitro showed high expression of syncytin-1, but no plac1 expression, demonstrating that trophoblast cell EVs express syncytin-1 on their surface. Placental alkaline phosphatase also showed high expression on BeWo EVs, but due to concern for cross reactivity to highly prevalent isoforms of intestinal and bone alkaline phosphatase, we utilized syncytin-1 as a marker for STEVs. In vivo, syncytin-1 protein expression was confirmed in maternal plasma EVs from Control and Preeclampsia subjects by Western blot, and overall, lower expression was noted in samples from patients with preeclampsia (n = 8). By nanoparticle analysis, EV profiles from Control and Preeclampsia groups showed similar total plasma EV quantities (p = 0.313) and size distribution (p = 0.415), but STEV quantitative signal, marked by syncytin-1 specific EVs, was significantly decreased in the Preeclampsia group (p = 2.8 × 10-11). Receiver operating characteristic curve demonstrated that STEV signal threshold cut-off of <0.316 was 95.2% sensitive and 95.6% specific for diagnosis of preeclampsia in this cohort (area under curve = 0.975 ± 0.020). In conclusion, we report that the syncytin-1 expressing EV profiles in maternal plasma might serve as a placental tissue specific biomarker for preeclampsia. (hide) EV-METRIC 45% (78th 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 Pregnant 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 Size-exclusion chromatography (non-commercial) UF (d)(U)C Protein markers EV: TSG101/ CD63/ Syncytin-1, PLAP/ Flotillin1 non-EV: Cytochrome C 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 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type Not specified Pelleting: speed (g) 120000 Ultra filtration Cut-off size (kDa) 100 Kda Membrane type Not specified Size-exclusion chromatography Total column volume (mL) 10 Sample volume/column (mL) 0.25 Resin type Sepharose 2B Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? No Detected EV-associated proteins Flotillin1/ CD63/ TSG101/ Syncytin-1/ PLAP Not detected contaminants Cytochrome C Fluorescent NTA Relevant measurements variables specified? NA Antibody details provided? No Detected EV-associated proteins Syncytin-1 Characterization: RNA analysis RNA analysis Type (RT)(q)PCR Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 50-100nm EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 5.5E6 EM EM-type Cryo-EM Image type Close-up

	EV200132	1/3	Homo sapiens	Blood plasma	(d)(U)C DG	James-Allan, Laura B	2020	45%
Study summary Full title Regulation of glucose homeostasis by small extracellular vesicles in normal pregnancy and in gestational diabetes All authors Laura B James-Allan, Frederick J Rosario, Kelsey Barner, Andrew Lai, Dominic Guanzon, H David McIntyre, Martha Lappas, Theresa L Powell, Carlos Salomon, Thomas Jansson Journal FASEB J Abstract The mechanisms underpinning maternal metabolic adaptations to a healthy pregnancy and in gestational (show more...)The mechanisms underpinning maternal metabolic adaptations to a healthy pregnancy and in gestational diabetes mellitus (GDM) remain poorly understood. We hypothesized that small extracellular vesicles (sEVs) isolated from healthy pregnant women promote islet glucose-stimulated insulin secretion (GSIS) and peripheral insulin resistance in nonpregnant mice and that sEVs from GDM women fail to stimulate insulin secretion and cause exacerbated insulin resistance. Small EVs were isolated from plasma of nonpregnant, healthy pregnant, and GDM women at 24-28 weeks of gestation. We developed a novel approach in nonpregnant mice involving a mini-osmotic pump for continuous 4-day jugular venous infusion of sEVs and determined their effects on glucose tolerance in vivo and islets and skeletal muscle in vitro. Fasting insulin was elevated in mice infused with pregnant sEVs as compared to sEVs from nonpregnant and GDM women. Mice infused with sEVs from GDM women developed glucose intolerance. GSIS was increased in mice infused with healthy pregnancy sEVs compared to mice receiving nonpregnant sEVs. GSIS and muscle basal insulin signaling, and insulin responsiveness were attenuated in mice infused with GDM sEVs. sEVs represent a novel mechanism regulating maternal glucose homeostasis in pregnancy and we speculate that altered sEV content contributes to the development of GDM. (hide) EV-METRIC 45% (78th 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 vesicles Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Density gradient Protein markers EV: TSG101/ PLAP/ CD63/ CD9 non-EV: Apolipoprotein B/ Argonaute2/ Grp94 Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Blood plasma Separation Method Density gradient Type Discontinuous Orientation Top-down Pelleting: duration (min) 120 Pelleting: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? Yes Detected EV-associated proteins CD9/ CD63/ TSG101 Not detected contaminants Grp94/ Apolipoprotein B/ Argonaute2 Fluorescent NTA Antibody details provided? No Detected EV-associated proteins PLAP/ CD63 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 1-1000 EV concentration Yes EM EM-type Transmission-EM Image type Close-up Report size (nm) 100

	EV200085	1/5	Rattus norvegicus	Primary Hippocampus neurons	DG (d)(U)C	Rohit Kumar	2020	45%
Study summary Full title Fibroblast Growth Factor 2-Mediated Regulation of Neuronal Exosome Release Depends on VAMP3/Cellubrevin in Hippocampal Neurons All authors Rohit Kumar, Qilin Tang, Stephan A Müller, Pan Gao, Diana Mahlstedt, Sofia Zampagni, Yi Tan, Andreas Klingl, Kai Bötzel, Stefan F Lichtenthaler, Günter U Höglinger, Thomas Koeglsperger Journal Advanced Science Abstract Extracellular vesicles (EVs) are endogenous membrane-derived vesicles that shuttle bioactive molecul (show more...)Extracellular vesicles (EVs) are endogenous membrane-derived vesicles that shuttle bioactive molecules between glia and neurons, thereby promoting neuronal survival and plasticity in the central nervous system (CNS) and contributing to neurodegenerative conditions. Although EVs hold great potential as CNS theranostic nanocarriers, the specific molecular factors that regulate neuronal EV uptake and release are currently unknown. A combination of patch-clamp electrophysiology and pH-sensitive dye imaging is used to examine stimulus-evoked EV release in individual neurons in real time. Whereas spontaneous electrical activity and the application of a high-frequency stimulus induce a slow and prolonged fusion of multivesicular bodies (MVBs) with the plasma membrane (PM) in a subset of cells, the neurotrophic factor basic fibroblast growth factor (bFGF) greatly increases the rate of stimulus-evoked MVB-PM fusion events and, consequently, the abundance of EVs in the culture medium. Proteomic analysis of neuronal EVs demonstrates bFGF increases the abundance of the v-SNARE vesicle-associated membrane protein 3 (VAMP3, cellubrevin) on EVs. Conversely, knocking-down VAMP3 in cultured neurons attenuates the effect of bFGF on EV release. The results determine the temporal characteristics of MVB-PM fusion in hippocampal neurons and reveal a new function for bFGF signaling in controlling neuronal EV release. (hide) EV-METRIC 45% (86th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin CD63-pHluorin transduced Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture DG (d)(U)C Protein markers EV: Alix/ CD81/ Flotillin1/ CD9/ GFP/ VAMP3/ VAMP2 non-EV: None Proteomics yes EV density (g/ml) 1.08-1.14 Show all info Study aim Function/Biogenesis/cargo sorting/Identification of content (omics approaches) Sample Species Rattus norvegicus Sample Type Cell culture supernatant EV-producing cells Primary Hippocampus neurons EV-harvesting Medium Serum free Cell viability (%) NA Cell count 500000 cells 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 MLA-80 Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 1 Wash: time (min) 90 Wash: Rotor Type TLA-55 Wash: speed (g) 100000 Density gradient Only used for validation of main results Yes Type Continuous Lowest density fraction 10% Highest density fraction 30% Total gradient volume, incl. sample (mL) 5.5 Sample volume (mL) 3 Orientation Bottom-up Rotor type SW 55 Ti Speed (g) 350000 Duration (min) 60 Fraction volume (mL) 0.49 Fraction processing Centrifugation Pelleting: volume per fraction 3 Pelleting: duration (min) 30 Pelleting: rotor type TLA-110 Pelleting: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? Yes Detected EV-associated proteins Alix/ CD81/ Flotillin1/ CD9/ GFP/ VAMP3 Not detected EV-associated proteins VAMP2 Proteomics database Yes Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 0-500 EV concentration Yes

	EV230020	1/9	Homo sapiens	ATCC PCS-500-012	(d)(U)C	Wang J	2020	44%
Study summary Full title Boosting the Biogenesis and Secretion of Mesenchymal Stem Cell-Derived Exosomes. All authors Wang J, Bonacquisti EE, Brown AD, Nguyen J Journal Cells Abstract A limitation of using exosomes to their fullest potential is their limited secretion from cells, a m (show more...)A limitation of using exosomes to their fullest potential is their limited secretion from cells, a major bottleneck to efficient exosome production and application. This is especially true for mesenchymal stem cells (MSCs), which can self-renew but have a limited expansion capacity, undergoing senescence after only a few passages, with exosomes derived from senescent stem cells showing impaired regenerative capacity compared to young cells. Here, we examined the effects of small molecule modulators capable of enhancing exosome secretion from MSCs. The treatment of MSCs with a combination of N-methyldopamine and norepinephrine robustly increased exosome production by three-fold without altering the ability of the MSC exosomes to induce angiogenesis, polarize macrophages to an anti-inflammatory phenotype, or downregulate collagen expression. These small molecule modulators provide a promising means to increase exosome production by MSCs. (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 (Differential) (ultra)centrifugation Protein markers EV: CD9/ CD63 non-EV: None Proteomics no Show all info Study aim Biogenesis/cargo sorting Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells ATCC PCS-500-012 EV-harvesting Medium EV-depleted medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 100000 Wash: time (min) 70 Wash: Rotor Type SW 32 Ti Wash: speed (g) 100000 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 Characterization: Lipid analysis No Characterization: Particle analysis NTA EV concentration Yes EM EM-type Transmission-EM Image type Close-up

	EV230020	8/9	Homo sapiens	ATCC PCS-500-012	(d)(U)C	Wang J	2020	44%
Study summary Full title Boosting the Biogenesis and Secretion of Mesenchymal Stem Cell-Derived Exosomes. All authors Wang J, Bonacquisti EE, Brown AD, Nguyen J Journal Cells Abstract A limitation of using exosomes to their fullest potential is their limited secretion from cells, a m (show more...)A limitation of using exosomes to their fullest potential is their limited secretion from cells, a major bottleneck to efficient exosome production and application. This is especially true for mesenchymal stem cells (MSCs), which can self-renew but have a limited expansion capacity, undergoing senescence after only a few passages, with exosomes derived from senescent stem cells showing impaired regenerative capacity compared to young cells. Here, we examined the effects of small molecule modulators capable of enhancing exosome secretion from MSCs. The treatment of MSCs with a combination of N-methyldopamine and norepinephrine robustly increased exosome production by three-fold without altering the ability of the MSC exosomes to induce angiogenesis, polarize macrophages to an anti-inflammatory phenotype, or downregulate collagen expression. These small molecule modulators provide a promising means to increase exosome production by MSCs. (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 Norepinephrine + N-methyldopamine Focus vesicles exosome Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Protein markers EV: CD9/ CD63 non-EV: None Proteomics no Show all info Study aim Biogenesis/cargo sorting Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells ATCC PCS-500-012 EV-harvesting Medium EV-depleted medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 100000 Wash: time (min) 70 Wash: Rotor Type SW 32 Ti Wash: speed (g) 100000 Characterization: Protein analysis None Protein Concentration Method Bradford Characterization: Lipid analysis No Characterization: Particle analysis NTA EV concentration Yes EM EM-type Transmission-EM Image type Close-up

	EV220247	1/5	Homo sapiens	adipose-derived mesenchymal stem cells	(d)(U)C	Lu Y	2020	44%
Study summary Full title Extracellular vesicle-enclosed miR-486-5p mediates wound healing with adipose-derived stem cells by promoting angiogenesis. All authors Lu Y, Wen H, Huang J, Liao P, Liao H, Tu J, Zeng Y Journal J Cell Mol Med Abstract Adipose-derived stem cells (ASC) are said to have a pivotal role in wound healing. Specifically, ASC (show more...)Adipose-derived stem cells (ASC) are said to have a pivotal role in wound healing. Specifically, ASC-secreted extracellular vesicles (EV) carry diverse cargos such as microRNAs (miRNAs) to participate in the ASC-based therapies. Considering its effects, we aimed to investigate the role of ASC-EVs in the cutaneous wound healing accompanied with the study on the specific cargo-medicated effects on wound healing. Two full-thickness excisional skin wounds were created on mouse dorsum, and wound healing was recorded at the indicated time points followed by histological analysis and immunofluorescence staining for CD31 and α-SMA. Human skin fibroblasts (HSFs) and human microvascular endothelial cells (HMECs) were co-cultured with EVs isolated from ASC (ASC-EVs), respectively, followed by the evaluation of their viability and mobility using CCK-8, scratch test and transwell migration assays. Matrigel-based angiogenesis assays were performed to evaluate vessel-like tube formation by HMECs in vitro. ASC-EVs accelerated the healing of full-thickness skin wounds, increased re-epithelialization and reduced scar thickness whilst enhanced collagen synthesis and angiogenesis in murine models. However, miR-486-5p antagomir abrogated the ASC-EVs-induced effects. Intriguingly, miR-486-5p was found to be highly enriched in ASC-EVs, exhibiting an increase in viability and mobility of HSFs and HMECs and enhanced the angiogenic activities of HMECs. Notably, we also demonstrated that ASC-EVs-secreted miR-486-5p achieved the aforesaid effects through its target gene Sp5. Hence, our results suggest that miR-486-5p released by ASC-EVs could be a critical mediator to develop an ASC-based therapeutic strategy for wound healing. (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 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 Protein markers EV: TSG101/ CD63 non-EV: calnexin Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells adipose-derived mesenchymal stem cells 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 50,000 g and 100,000 g Pelleting performed Yes Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 70000 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 CD63/ TSG101 Not detected contaminants calnexin Characterization: RNA analysis RNA analysis Type (RT)(q)PCR Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 30-100 EM EM-type Transmission-EM Image type Close-up

	EV220243	1/5	Homo sapiens	Umbilical cord-derived mesenchymal stem cells	(d)(U)C Filtration	Cheng S	2020	44%
Study summary Full title Extracellular vesicle-carried microRNA-27b derived from mesenchymal stem cells accelerates cutaneous wound healing via E3 ubiquitin ligase ITCH. All authors Cheng S, Xi Z, Chen G, Liu K, Ma R, Zhou C Journal J Cell Mol Med Abstract Mesenchymal stem cells (MSCs) have been highlighted as promising candidate cells in relation to cuta (show more...)Mesenchymal stem cells (MSCs) have been highlighted as promising candidate cells in relation to cutaneous wound healing. The current study aimed to investigate whether MSC-derived extracellular vesicles (EVs) could transfer microRNA-27b (miR-27b) to influence cutaneous wound healing. The miR-27b expression was examined in the established cutaneous wound mouse model, and its correlation with the wound healing rate was evaluated by Pearson's correlation analysis. The identified human umbilical cord MSC-derived EVs were co-cultured with human immortal keratinocyte line HaCaT and human skin fibroblasts (HSFs). The mice with cutaneous wound received injections of MSC-derived EVs. The effects of EVs or miR-27b loaded on wound healing and cellular functions were analysed via gain- and loss-of-function approaches in the co-culture system. Dual-luciferase reporter gene assay was employed to verify the relationship between miR-27b and Itchy E3 ubiquitin protein ligase (ITCH). Rescue experiments were conducted to investigate the underlying mechanisms associated with the ITCH/JUNB/inositol-requiring enzyme 1α (IRE1α) axis. miR-27b was down-regulated in the mouse model, with its expression found to be positively correlated with the wound healing rate. Abundant miR-27b was detected in the MSC-derived EVs, while EV-transferred miR-27b improved cutaneous wound healing in mice and improved proliferation and migration of HaCaT cells and HSFs in vitro. As a target of miR-27b, ITCH was found to repress cell proliferation and migration. ITCH enhanced the JUNB ubiquitination and degradation, ultimately inhibiting JUNB and IRE1α expressions and restraining wound healing. Collectively, MSC-derived EVs transferring miR-27b can promote cutaneous wound healing via ITCH/JUNB/IRE1α signalling, providing insight with clinical implications. (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 extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Filtration Protein markers EV: TSG101/ CD81/ CD63 non-EV: Calnexin Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Umbilical cord-derived mesenchymal stem cells EV-harvesting Medium EV-depleted medium Preparation of EDS >=18h at >= 100,000g Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type Type 70 Ti Pelleting: speed (g) 110000 Wash: time (min) 70 Wash: Rotor Type Type 70 Ti Wash: speed (g) 110000 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Detected EV-associated proteins CD63/ TSG101/ CD81 Not detected contaminants Calnexin Characterization: Lipid analysis No Characterization: Particle analysis DLS Report type Size range/distribution Reported size (nm) 30-100 EM EM-type Transmission-EM Image type Close-up

	EV220237	1/2	Homo sapiens	bone marrow-derived mesenchymal stem cells	(d)(U)C Filtration	Wu D	2020	44%
Study summary Full title Exosomes Derived from Bone Mesenchymal Stem Cells with the Stimulation of FeO Nanoparticles and Static Magnetic Field Enhance Wound Healing Through Upregulated miR-21-5p. All authors Wu D, Kang L, Tian J, Wu Y, Liu J, Li Z, Wu X, Huang Y, Gao B, Wang H, Wu Z, Qiu G Journal Int J Nanomedicine Abstract Both magnetic nanoparticles (MNPs) and exosomes derived from bone mesenchymal stem cells (BMSC-Exos) (show more...)Both magnetic nanoparticles (MNPs) and exosomes derived from bone mesenchymal stem cells (BMSC-Exos) have been reported to improve wound healing. In this study, novel exosomes (mag-BMSC-Exos) would be fabricated from BMSCs with the stimulation of MNPs and a static magnetic field (SMF) to further enhance wound repair. (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 (Differential) (ultra)centrifugation Filtration Protein markers EV: TSG101/ CD81/ CD63/ CD9 non-EV: calnexin Proteomics no Show all info Study aim Function/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 Serum-containing, but physical separation of serum EVs and secreted EVs (e.g. Bioreactor flask) 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: speed (g) 100000 Wash: time (min) 70 Wash: speed (g) 100000 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? No Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD9/ CD63/ TSG101/ CD81 Not detected contaminants calnexin Characterization: RNA analysis RNA analysis Type RNA sequencing Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 118.1 EV concentration Yes EM EM-type Transmission-EM Image type Close-up

	EV220237	2/2	Homo sapiens	bone marrow-derived mesenchymal stem cells	(d)(U)C Filtration	Wu D	2020	44%
Study summary Full title Exosomes Derived from Bone Mesenchymal Stem Cells with the Stimulation of FeO Nanoparticles and Static Magnetic Field Enhance Wound Healing Through Upregulated miR-21-5p. All authors Wu D, Kang L, Tian J, Wu Y, Liu J, Li Z, Wu X, Huang Y, Gao B, Wang H, Wu Z, Qiu G Journal Int J Nanomedicine Abstract Both magnetic nanoparticles (MNPs) and exosomes derived from bone mesenchymal stem cells (BMSC-Exos) (show more...)Both magnetic nanoparticles (MNPs) and exosomes derived from bone mesenchymal stem cells (BMSC-Exos) have been reported to improve wound healing. In this study, novel exosomes (mag-BMSC-Exos) would be fabricated from BMSCs with the stimulation of MNPs and a static magnetic field (SMF) to further enhance wound repair. (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 Fe3O4 (50g/ml)-incubated static magnetic field (100mT) Focus vesicles exosome Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Filtration Protein markers EV: TSG101/ CD81/ CD63/ CD9 non-EV: calnexin Proteomics no Show all info Study aim Function/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 Serum-containing, but physical separation of serum EVs and secreted EVs (e.g. Bioreactor flask) 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: speed (g) 100000 Wash: time (min) 70 Wash: speed (g) 100000 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? No Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD9/ CD63/ TSG101/ CD81 Not detected contaminants calnexin Characterization: RNA analysis RNA analysis Type RNA sequencing Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 116.2 EV concentration Yes EM EM-type Transmission-EM Image type Close-up

	EV210348	2/6	Homo sapiens	Serum	(d)(U)C DC	Arya R	2020	44%
Study summary Full title Serum Small Extracellular Vesicles Proteome of Tuberculosis Patients Demonstrated Deregulated Immune Response. All authors Arya R, Dabral D, Faruquee HM, Mazumdar H, Patgiri SJ, Deka T, Basumatary R, Kupa RU, Semy C, Kapfo W, Liegise K, Kaur I, Choedon T, Kumar P, Behera RK, Deori P, Nath R, Khalo K, Saikia L, Khamo V, Nanda RK Journal Proteomics Clin Appl Abstract Detailed understanding of host pathogen interaction in tuberculosis is an important avenue for ident (show more...)Detailed understanding of host pathogen interaction in tuberculosis is an important avenue for identifying novel therapeutic targets. Small extracellular vesicles (EVs) like exosomes that are rich in proteins, nucleic acids and lipids, act as messengers and may show altered composition in disease conditions. (hide) EV-METRIC 44% (86th 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 (Differential) (ultra)centrifugation Density cushion Protein markers EV: KYAT3/ SERPINA1/ HP/ APOC3 non-EV: None Proteomics no Show all info Study aim Identification of content (omics approaches) 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 No Density cushion Density medium Sucrose Sample volume Not specified Cushion volume Not specified Centrifugation time 90 Centrifugation speed 110000 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 KYAT3/ SERPINA1/ HP/ APOC3 Characterization: Lipid analysis No Characterization: Particle analysis DLS Report type Size range/distribution EM EM-type Immuno-EM EM protein CD9 Image type Close-up Report size (nm) 20-200

	EV210348	4/6	Homo sapiens	Serum	(d)(U)C DC	Arya R	2020	44%
Study summary Full title Serum Small Extracellular Vesicles Proteome of Tuberculosis Patients Demonstrated Deregulated Immune Response. All authors Arya R, Dabral D, Faruquee HM, Mazumdar H, Patgiri SJ, Deka T, Basumatary R, Kupa RU, Semy C, Kapfo W, Liegise K, Kaur I, Choedon T, Kumar P, Behera RK, Deori P, Nath R, Khalo K, Saikia L, Khamo V, Nanda RK Journal Proteomics Clin Appl Abstract Detailed understanding of host pathogen interaction in tuberculosis is an important avenue for ident (show more...)Detailed understanding of host pathogen interaction in tuberculosis is an important avenue for identifying novel therapeutic targets. Small extracellular vesicles (EVs) like exosomes that are rich in proteins, nucleic acids and lipids, act as messengers and may show altered composition in disease conditions. (hide) EV-METRIC 44% (86th 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 Drug naive active tuberculosis Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Density cushion Protein markers EV: KYAT3/ SERPINA1/ HP/ APOC3 non-EV: None Proteomics no Show all info Study aim Identification of content (omics approaches) 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 No Density cushion Density medium Sucrose Sample volume Not specified Cushion volume Not specified Centrifugation time 90 Centrifugation speed 110000 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 KYAT3/ SERPINA1/ HP/ APOC3 Characterization: Lipid analysis No Characterization: Particle analysis DLS Report type Size range/distribution EM EM-type Immuno-EM EM protein CD9 Image type Close-up Report size (nm) 20-200

	EV210348	6/6	Homo sapiens	Serum	(d)(U)C DC	Arya R	2020	44%
Study summary Full title Serum Small Extracellular Vesicles Proteome of Tuberculosis Patients Demonstrated Deregulated Immune Response. All authors Arya R, Dabral D, Faruquee HM, Mazumdar H, Patgiri SJ, Deka T, Basumatary R, Kupa RU, Semy C, Kapfo W, Liegise K, Kaur I, Choedon T, Kumar P, Behera RK, Deori P, Nath R, Khalo K, Saikia L, Khamo V, Nanda RK Journal Proteomics Clin Appl Abstract Detailed understanding of host pathogen interaction in tuberculosis is an important avenue for ident (show more...)Detailed understanding of host pathogen interaction in tuberculosis is an important avenue for identifying novel therapeutic targets. Small extracellular vesicles (EVs) like exosomes that are rich in proteins, nucleic acids and lipids, act as messengers and may show altered composition in disease conditions. (hide) EV-METRIC 44% (86th 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 Non-tuberculosis Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Density cushion Protein markers EV: KYAT3/ SERPINA1/ HP/ APOC3 non-EV: None Proteomics no Show all info Study aim Identification of content (omics approaches) 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 No Density cushion Density medium Sucrose Sample volume Not specified Cushion volume Not specified Centrifugation time 90 Centrifugation speed 110000 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 KYAT3/ SERPINA1/ HP/ APOC3 Characterization: Lipid analysis No Characterization: Particle analysis DLS Report type Size range/distribution EM EM-type Immuno-EM EM protein CD9 Image type Close-up Report size (nm) 20-200

	EV210325	1/3	Homo sapiens	MDAMB231	(d)(U)C Filtration	Han S	2020	44%
Study summary Full title Isolation and analysis of extracellular vesicles in a Morpho butterfly wing-integrated microvortex biochip. All authors Han S, Xu Y, Sun J, Liu Y, Zhao Y, Tao W, Chai R Journal Biosens Bioelectron Abstract With the function of mediating intercellular communication between cells, extracellular vesicles (EV (show more...)With the function of mediating intercellular communication between cells, extracellular vesicles (EVs) have been intently studied for their physiopathology and clinical application values. However, efficient EV isolation from biological fluids remains a significant challenge. To address this, this work constructs a new microvortex chip that can isolate EVs efficiently by integrating the lipid nanoprobe modified Morpho Menelaus (M. Menelaus) butterfly wing into microfluidic chip. M. Menelaus wing is well known for its orderly arranged periodic nanostructures and can generate microvortex when liquid passes through it, leading to increased interaction between EVs and M. Menelaus wing. In addition, the nanoprobe containing lipid tails can be inserted into EVs through their lipid bilayer membrane structure. Based on the described properties, high-throughput enrichment of EVs with over 70% isolation efficiency was realized. Moreover, it was demonstrated that the nanoprobe system based on M. Menelaus wing enabled downstream biological analysis of nucleic acids and proteins in EVs. Microvortex chips showed potential application value in efficient EV isolation for biomedical research and cancer diagnosis. (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 overexpressing of GPC1 mRNA Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Filtration Protein markers EV: CD9/ CD63/ Flotillin1 non-EV: None Proteomics no 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 MDAMB231 EV-harvesting Medium Serum free medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: speed (g) 100000 Wash: time (min) 70 Wash: speed (g) 100000 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 CD9/ CD63/ Flotillin1 Characterization: RNA analysis RNA analysis Type (RT)(q)PCR Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 0-400 EV concentration Yes EM EM-type Transmission-EM Image type Wide-field Report size (nm) 20-200

	EV210325	2/3	Homo sapiens	MDAMB231	Morpho butterfly wing-integrated microvortex biochip	Han S	2020	44%
Study summary Full title Isolation and analysis of extracellular vesicles in a Morpho butterfly wing-integrated microvortex biochip. All authors Han S, Xu Y, Sun J, Liu Y, Zhao Y, Tao W, Chai R Journal Biosens Bioelectron Abstract With the function of mediating intercellular communication between cells, extracellular vesicles (EV (show more...)With the function of mediating intercellular communication between cells, extracellular vesicles (EVs) have been intently studied for their physiopathology and clinical application values. However, efficient EV isolation from biological fluids remains a significant challenge. To address this, this work constructs a new microvortex chip that can isolate EVs efficiently by integrating the lipid nanoprobe modified Morpho Menelaus (M. Menelaus) butterfly wing into microfluidic chip. M. Menelaus wing is well known for its orderly arranged periodic nanostructures and can generate microvortex when liquid passes through it, leading to increased interaction between EVs and M. Menelaus wing. In addition, the nanoprobe containing lipid tails can be inserted into EVs through their lipid bilayer membrane structure. Based on the described properties, high-throughput enrichment of EVs with over 70% isolation efficiency was realized. Moreover, it was demonstrated that the nanoprobe system based on M. Menelaus wing enabled downstream biological analysis of nucleic acids and proteins in EVs. Microvortex chips showed potential application value in efficient EV isolation for biomedical research and cancer diagnosis. (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 overexpressing of GPC1 mRNA 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 Morpho butterfly wing-integrated microvortex biochip Protein markers EV: CD9/ CD63/ Flotillin1 non-EV: None Proteomics no 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 MDAMB231 EV-harvesting Medium Serum free medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: speed (g) 100000 Wash: time (min) 70 Wash: speed (g) 100000 Other Name other separation method Morpho butterfly wing-integrated microvortex biochip Other Name other separation method 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 CD9/ CD63/ Flotillin1 Characterization: RNA analysis RNA analysis Type (RT)(q)PCR Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 0-400 EV concentration Yes EM EM-type Transmission-EM Image type Wide-field Report size (nm) 20-200

	EV210228	1/4	Homo sapiens	Blood plasma	(d)(U)C Filtration	Tronco, Júlia A	2020	44%
Study summary Full title Alpha-2-macroglobulin from circulating exosome-like vesicles is increased in women with preterm pregnancies All authors Júlia A Tronco, Bruna R de A Ramos, Natália M Bastos, Sérgio A Alcântara, Juliano C da Silveira, Márcia G da Silva Journal Sci Rep Abstract Preterm labor (PTL) and Preterm Premature Rupture of Membranes (PPROM) impose substantial morbimorta (show more...)Preterm labor (PTL) and Preterm Premature Rupture of Membranes (PPROM) impose substantial morbimortality on mothers and newborns. Exosomes act in intercellular communication carrying molecules involved in physiopathological processes. Little is known about exosomal proteins in prematurity. Our aim was to evaluate the protein expression of hemopexin, C1 inhibitor (C1INH) and alpha-2-macroglobulin (A2M) from circulating exosomes of women with PTL and PPROM. Plasma was obtained from PTL, PPROM, Term in labor and Term out of labor (T) patients, exosomes were isolated by ultracentrifugation, then lysed and the proteins quantified. Western Blot (WB) and Nanoparticle Tracking Analysis (NTA) were performed. Data were compared by Kruskal-Wallis, unpaired T-test and one-way ANOVA. WB and NTA confirmed exosome isolation (concentration: 4.3 × 1010 particles/ml ± 1.9 × 1010). There was no difference regarding hemopexin or C1INH expression between the groups. For A2M, the fold change was significantly higher on preterm groups when compared to term groups (1.07 ± 0.30 vs. 0.42 ± 0.17, p < 0.0001). Higher levels of A2M in circulating exosomes are linked to preterm pregnancies. sEV are strong candidates to intermediate maternal-fetal communication, carrying preterm labor-related immunomodulatory proteins. (hide) EV-METRIC 44% (77th 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 Healthy pregnant (at term but not in labour) 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: CD9/ CD63/ Hemopexin/ C1INH/ A2M non-EV: Cytochrome C Proteomics no Show all info Study aim Function/Biomarker Sample Species Homo sapiens Sample Type Blood plasma Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 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) 120000 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? No Detected EV-associated proteins CD9/ CD63/ Hemopexin/ C1INH/ A2M Not detected contaminants Cytochrome C Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 173.2 EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 4.12x10E10 EM EM-type Transmission-EM Image type Wide-field Report size (nm) >200nm

				201 - 250 of 1113

Study summary

Study data

Study summary

Study data

Study summary

Study data

Study summary

Study data

Study summary

Study data

Study summary

Study data

Study summary

Study data

Study summary

Study data

Study summary

Study data

Study summary

Study data

Study summary

Study data

Study summary

Study data

Study summary

Study data

Study summary

Study data

Study summary

Study data

Study summary

Study data

Study summary

Study data

Study summary

Study data

Study summary

Study data

Study summary

Study data

Study summary

Study data

Study summary

Study data

Study summary

Study data

Study summary

Study data

Study summary

Study data

Study summary

Study data

Study summary

Study data

Study summary

Study data

Study summary

Study data

Study summary

Study data

Study summary

Study data

Study summary

Study data

Study summary

Study data

Study summary

Study data

Study summary

Study data

Study summary

Study data

Study summary

Study data

Study summary

Study data

Study summary

Study data

Study summary

Study data