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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV210279	1/2	Homo sapiens	A431	(d)(U)C DG	Libregts SFWM	2018	63%
Study summary Full title Flow cytometric analysis of extracellular vesicle subsets in plasma: impact of swarm by particles of non-interest. All authors Libregts SFWM, Arkesteijn GJA, Németh A, Nolte-'t Hoen ENM, Wauben MHM Journal J Thromb Haemost Abstract Essentials Extracellular vesicles (EVs) in biological fluids are promising biomarkers for disease. F (show more...)Essentials Extracellular vesicles (EVs) in biological fluids are promising biomarkers for disease. Fluorescence-based flow cytometric analysis is suitable to detect low abundant EV subsets. Particles of non-interest can induce false-positive light scatter and fluorescent signals. Interference of particles of non-interest can be monitored by analyzing serial dilutions. (hide) EV-METRIC 63% (93rd percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Density gradient Protein markers EV: EGFR/ CD9/ CD142 non-EV: None Proteomics no Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells A431 EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type SW28 or SW32 Pelleting: speed (g) 100000 Density gradient Type Continuous Lowest density fraction 0.4M Highest density fraction 2.08M Total gradient volume, incl. sample (mL) 11.6 Sample volume (mL) 0.3 Orientation Bottom-up Rotor type SW 40 Ti Speed (g) 270000 Duration (min) 960 Fraction volume (mL) 1 Fraction processing None Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Flow cytometry Type of Flow cytometry BD Influx/ Beckman Coulter CytoFLEX LX Hardware adaptation to ~100nm EV's BD Influx: Adapted small particle detector Calibration bead size 0.1 and 0.2 Detected EV-associated proteins CD9/ EGFR/ CD142 Characterization: Lipid analysis No Characterization: Particle analysis Particle analysis: flow cytometry Flow cytometer type BD Influx/ Beckman Coulter Cytoflex LX Hardware adjustment BD Influx: adapted small particle detector Calibration bead size 0.1 and 0.2 EV concentration Yes

	EV210279	2/2	Homo sapiens	Blood plasma	qEV column	Libregts SFWM	2018	13%
Study summary Full title Flow cytometric analysis of extracellular vesicle subsets in plasma: impact of swarm by particles of non-interest. All authors Libregts SFWM, Arkesteijn GJA, Németh A, Nolte-'t Hoen ENM, Wauben MHM Journal J Thromb Haemost Abstract Essentials Extracellular vesicles (EVs) in biological fluids are promising biomarkers for disease. F (show more...)Essentials Extracellular vesicles (EVs) in biological fluids are promising biomarkers for disease. Fluorescence-based flow cytometric analysis is suitable to detect low abundant EV subsets. Particles of non-interest can induce false-positive light scatter and fluorescent signals. Interference of particles of non-interest can be monitored by analyzing serial dilutions. (hide) EV-METRIC 13% (29th 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 Commercial method Protein markers EV: CD63/ CD9 non-EV: None 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 qEV column Characterization: Protein analysis Protein Concentration Method Other/ Absorbance spectrometry (280 nm) Western Blot Detected EV-associated proteins CD9/ CD63 Characterization: Lipid analysis No Characterization: Particle analysis Particle analysis: flow cytometry Flow cytometer type BD Influx/ Beckman Coulter Cytoflex LX Hardware adjustment BD Influx: adapted small particle detector Calibration bead size 0.1 and 0.2 EV concentration Yes

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EV-TRACK ID	EV210279
species	Homo sapiens
sample type	Cell culture	Blood plasma
cell type	A431	NA
medium	EV-depleted medium	NA
condition	Control condition	Control condition
separation protocol	dUC/ Density gradient	qEV column
Exp. nr.	1	2
EV-METRIC %	63	13