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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV170001	1/1	Homo sapiens	MCF7 Rab27b-GFP	DG (d)(U)C Filtration UF	Vergauwen, Glenn	2017	100%
Study summary Full title Confounding factors of ultrafiltration and protein analysis in extracellular vesicle research. All authors Vergauwen G, Dhondt B, Van Deun J, De Smedt E, Berx G, Timmerman E, Gevaert K, Miinalainen I, Cocquyt V, Braems G, Van den Broecke R, Denys H, De Wever O, Hendrix A. Journal Sci Rep Abstract Identification and validation of extracellular vesicle (EV)-associated biomarkers requires robust is (show more...)Identification and validation of extracellular vesicle (EV)-associated biomarkers requires robust isolation and characterization protocols. We assessed the impact of some commonly implemented pre-analytical, analytical and post-analytical variables in EV research. Centrifugal filters with different membrane types and pore sizes are used to reduce large volume biofluids prior to EV isolation or to concentrate EVs. We compared five commonly reported filters for their efficiency when using plasma, urine and EV-spiked PBS. Regenerated cellulose membranes with pore size of 10 kDa recovered EVs the most efficient. Less than 40% recovery was achieved with other filters. Next, we analyzed the effect of the type of protein assays to measure EV protein in colorimetric and fluorometric kits. The fluorometric assay Qubit measured low concentration EV and BSA samples the most accurately with the lowest variation among technical and biological replicates. Lastly, we quantified Optiprep remnants in EV samples from density gradient ultracentrifugation and demonstrate that size-exclusion chromatography efficiently removes Optiprep from EVs. In conclusion, choice of centrifugal filters and protein assays confound EV analysis and should be carefully considered to increase efficiency towards biomarker discovery. SEC-based removal of Optiprep remnants from EVs can be considered for downstream applications. (hide) EV-METRIC 100% (99th 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 DG (d)(U)C Filtration UF Protein markers EV: Alix/ CD81/ TSG101 non-EV: PMP70/ Argonaute-2 Proteomics no EV density (g/ml) 1.094 Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells MCF7 Rab27b-GFP EV-harvesting Medium EV-depleted serum Preparation of EDS >=18h at >=100,000g Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Pelleting performed No Density gradient Only used for validation of main results Yes Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 16.5 Sample volume (mL) 1 Orientation Top-down Rotor type SW 32.1 Ti Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 16 Pelleting: duration (min) 180 Pelleting: rotor type SW 32.1 Ti Pelleting: speed (g) 100000 Filtration steps 0.45µm > x > 0.22µm, Ultra filtration Cut-off size (kDa) 10 Membrane type Regenerated cellulose Characterization: Protein analysis Protein Concentration Method Fluorometric assay (e.g. Qubit, NanoOrange,…) Western Blot Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins Alix/ CD81/ TSG101 Not detected contaminants Argonaute-2/ PMP70 Characterization: RNA analysis RNA analysis Type (RT)-(q)PCR Characterization: Particle analysis NTA Report type Modus Reported size (nm) 113 EM EM-type Transmission-EM Image type Close-up, Wide-field

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EV-TRACK ID	EV170001
species	Homo sapiens
sample type	Cell culture
cell type	MCF7 Rab27b-GFP
condition	Control condition
separation protocol	DG (d)(U)C Filtration UF
Exp. nr.	1
EV-METRIC %	100