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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV140107	1/1	Homo sapiens	Platelet supernatant	(d)(U)C	Böing AN	2014	25%
Study summary Full title Single-step isolation of extracellular vesicles by size-exclusion chromatography All authors Böing AN, van der Pol E, Grootemaat AE, Coumans FA, Sturk A, Nieuwland R Journal J Extracell Vesicles Abstract BACKGROUND: Isolation of extracellular vesicles from plasma is a challenge due to the presence of pr (show more...)BACKGROUND: Isolation of extracellular vesicles from plasma is a challenge due to the presence of proteins and lipoproteins. Isolation of vesicles using differential centrifugation or density-gradient ultracentrifugation results in co-isolation of contaminants such as protein aggregates and incomplete separation of vesicles from lipoproteins, respectively. AIM: To develop a single-step protocol to isolate vesicles from human body fluids. METHODS: Platelet-free supernatant, derived from platelet concentrates, was loaded on a sepharose CL-2B column to perform size-exclusion chromatography (SEC; n=3). Fractions were collected and analysed by nanoparticle tracking analysis, resistive pulse sensing, flow cytometry and transmission electron microscopy. The concentrations of high-density lipoprotein cholesterol (HDL) and protein were measured in each fraction. RESULTS: Fractions 9-12 contained the highest concentrations of particles larger than 70 nm and platelet-derived vesicles (46%±6 and 61%±2 of totals present in all collected fractions, respectively), but less than 5% of HDL and less than 1% of protein (4.8%±1 and 0.65%±0.3, respectively). HDL was present mainly in fractions 18-20 (32%±2 of total), and protein in fractions 19-21 (36%±2 of total). Compared to the starting material, recovery of platelet-derived vesicles was 43%±23 in fractions 9-12, with an 8-fold and 70-fold enrichment compared to HDL and protein. CONCLUSIONS: SEC efficiently isolates extracellular vesicles with a diameter larger than 70 nm from platelet-free supernatant of platelet concentrates. Application SEC will improve studies on the dimensional, structural and functional properties of extracellular vesicles. (hide) EV-METRIC 25% (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 Platelet supernatant Sample origin NAY 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 (d)(U)C Protein markers EV: CD63/ CD9 non-EV: Proteomics no Show all info Study aim Technical Sample Species Homo sapiens Sample Type Platelet supernatant Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Pelleting performed No Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins CD63/ CD9 Characterization: Particle analysis NTA TRPS EM EM-type transmission EM Image type Wide-field

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EV-TRACK ID	EV140107
species	Homo sapiens
sample type	Platelet supernatant
condition	NAY
separation protocol	(d)(U)C
Exp. nr.	1
EV-METRIC %	25