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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV200037	1/2	Homo sapiens	HLA-DR15+ B cells	DG SEC (non-commercial) PEG precipitation	Xiaogang Zhang	2020	63%
Study summary Full title A novel three step protocol to isolate extracellular vesicles from plasma or cell culture medium with both high yield and purity All authors Xiaogang Zhang , Ellen G. F. Borg , A. Manuel Liaci , Harmjan R. Vos & Willem Stoorvogel Journal J Extracell Vesicles Abstract Extracellular vesicles (EV) are membrane encapsulated nanoparticles that can function in intercellul (show more...)Extracellular vesicles (EV) are membrane encapsulated nanoparticles that can function in intercellular communication, and their presence in biofluids can be indicative for (patho)physiological conditions. Studies aiming to resolve functionalities of EV or to discover EV-associated biomarkers for disease in liquid biopsies are hampered by limitations of current protocols to isolate EV from biofluids or cell culture medium. EV isolation is complicated by the >105-fold numerical excess of other types of particles, including lipoproteins and protein complexes. In addition to persisting contaminants, currently available EV isolation methods may suffer from inefficient EV recovery, bias for EV subtypes, interference with the integrity of EV membranes, and loss of EV functionality. In this study, we established a novel three-step non-selective method to isolate EV from blood or cell culture media with both high yield and purity, resulting in 71% recovery and near to complete elimination of unrelated (lipo)proteins. This EV isolation procedure is independent of ill-defined commercial kits, and apart from an ultracentrifuge, does not require specialised expensive equipment. (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 Density gradient Size-exclusion chromatography (non-commercial) PEG precipitation Protein markers EV: CD81/ MHC2/ CD63 non-EV: None Proteomics no EV density (g/ml) 1.09-1.13 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 HLA-DR15+ B cells EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g Cell viability (%) 98 Separation Method Density gradient Type Continuous Lowest density fraction 0% Highest density fraction 60% Total gradient volume, incl. sample (mL) 4 Sample volume (mL) 2 Orientation Bottom-up Rotor type SW 60 Ti Speed (g) 200000 Duration (min) 960 Fraction volume (mL) 0.3 Fraction processing Size-exclusion chromatography Size-exclusion chromatography Total column volume (mL) 10 Sample volume/column (mL) 0.6 Resin type Sepharose CL-2B Other Name other separation method PEG precipitation 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 CD63/ MHC2/ CD81 Characterization: Lipid analysis No EM EM-type Transmission-EM Image type Wide-field

	EV200037	2/2	Homo sapiens	Blood plasma	DG SEC (non-commercial) PEG precipitation	Xiaogang Zhang	2020	63%
Study summary Full title A novel three step protocol to isolate extracellular vesicles from plasma or cell culture medium with both high yield and purity All authors Xiaogang Zhang , Ellen G. F. Borg , A. Manuel Liaci , Harmjan R. Vos & Willem Stoorvogel Journal J Extracell Vesicles Abstract Extracellular vesicles (EV) are membrane encapsulated nanoparticles that can function in intercellul (show more...)Extracellular vesicles (EV) are membrane encapsulated nanoparticles that can function in intercellular communication, and their presence in biofluids can be indicative for (patho)physiological conditions. Studies aiming to resolve functionalities of EV or to discover EV-associated biomarkers for disease in liquid biopsies are hampered by limitations of current protocols to isolate EV from biofluids or cell culture medium. EV isolation is complicated by the >105-fold numerical excess of other types of particles, including lipoproteins and protein complexes. In addition to persisting contaminants, currently available EV isolation methods may suffer from inefficient EV recovery, bias for EV subtypes, interference with the integrity of EV membranes, and loss of EV functionality. In this study, we established a novel three-step non-selective method to isolate EV from blood or cell culture media with both high yield and purity, resulting in 71% recovery and near to complete elimination of unrelated (lipo)proteins. This EV isolation procedure is independent of ill-defined commercial kits, and apart from an ultracentrifuge, does not require specialised expensive equipment. (hide) EV-METRIC 63% (91st 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 Density gradient Size-exclusion chromatography (non-commercial) PEG precipitation Protein markers EV: CD81/ CD63/ CD9 non-EV: None Proteomics yes EV density (g/ml) 1.09-1.13 Show all info Study aim New methodological development/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Blood plasma Separation Method Density gradient Type Continuous Lowest density fraction 0% Highest density fraction 60% Total gradient volume, incl. sample (mL) 4 Sample volume (mL) 2 Orientation Bottom-up Rotor type SW 60 Ti Speed (g) 200000 Duration (min) 960 Fraction volume (mL) 0.3 Fraction processing Size-exclusion chromatography Size-exclusion chromatography Total column volume (mL) 10 Sample volume/column (mL) 0.6 Resin type Sepharose CL-2B Other Name other separation method PEG precipitation 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 CD9/ CD63/ CD81 Proteomics database Yes: Characterization: Lipid analysis No EM EM-type Transmission-EM/ Cryo-EM Image type Wide-field

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EV-TRACK ID	EV200037
species	Homo sapiens
sample type	Cell culture	Blood plasma
cell type	HLA-DR15+ B cells	NA
medium	EV-depleted medium	NA
condition	Control condition	Control condition
separation protocol	Density gradient Size-exclusion chromatography (non-commercial) PEG precipitation	Density gradient Size-exclusion chromatography (non-commercial) PEG precipitation
Exp. nr.	1	2
EV-METRIC %	63	63