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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Isolation protocol	First author	Year	EV-METRIC
	EV170028	1/1	Homo sapiens	Cell culture supernatant	Commercial method	Foers AD	2017	77%
Study summary Full title Enrichment of extracellular vesicles from human synovial fluid using size exclusion chromatography. All authors Foers AD, Chatfield S, Dagley LF, Scicluna BJ, Webb AI, Cheng L, Hill AF, Wicks IP, Pang KC. Journal J Extracell Vesicles Abstract As a complex biological fluid, human synovial fluid (SF) presents challenges for extracellular vesic (show more...)As a complex biological fluid, human synovial fluid (SF) presents challenges for extracellular vesicle (EV) enrichment using standard methods. In this study of human SF, a size exclusion chromatography (SEC)-based method of EV enrichment is shown to deplete contaminants that remain after standard ultracentrifugation-based enrichment methods. Specifically, considerable levels of serum albumin, the high-density lipoprotein marker, apolipoprotein A-I, fibronectin and other extracellular proteins and debris are present in EVs prepared by differential ultracentrifugation. While the addition of a sucrose density gradient purification step improved purification quality, some contamination remained. In contrast, using a SEC-based approach, SF EVs were efficiently separated from serum albumin, apolipoprotein A-I and additional contaminating proteins that co-purified with high-speed centrifugation. Finally, using high-resolution mass spectrometry analysis, we found that residual contaminants which remain after SEC, such as fibronectin and other extracellular proteins, can be successfully depleted by proteinase K. Taken together, our results highlight the limitations of ultracentrifugation-based methods of EV isolation from complex biological fluids and suggest that SEC can be used to obtain higher purity EV samples. In this way, SEC-based methods are likely to be useful for identifying EV-enriched components and improving understanding of EV function in disease. (hide) EV-METRIC 77% (98th 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 electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation 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 Focus vesicles exosome Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography Commercial method Adj. k-factor 88.25 (pelleting) / 88.25 (washing) Protein markers EV: Alix/ CD63/ CD81/ TSG101 non-EV: GM130/ cytochromec Proteomics no Show all info Study aim Function, Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant Sample Condition Colorectal cancer EV-producing cells Primary tumor-derived fibroblasts EV-harvesting Medium EV-depleted serum Preparation of EDS overnight (16h) at >=100,000g Isolation Method Differential ultracentrifugation 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: time(min) 70 Pelleting: rotor type Type 50.3 Ti Pelleting: speed (g) 100000 Pelleting: adjusted k-factor 88.25 Wash: time (min) 70 Wash: Rotor Type Type 50.3 Ti Wash: speed (g) 100000 Wash: adjusted k-factor 88.25 Characterization: Protein analysis Protein Concentration Method BCA Protein Concentration 100 Western Blot Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins Alix, CD63, CD81, TSG101 Not detected contaminants GM130, cytochrome c Fluorescent NTA Characterization: Particle analysis NTA Report type Modus Reported size (nm) 113 EV concentration Yes Particle yield 1.57E+12 particles/million cells EM EM-type Transmission-EM Image type Close-up, Wide-field

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