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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV160003	1/1	Mus musculus	DC2.4	(d)(U)C	Morales-Kastresana A	2016	14%
Study summary Full title Labeling Extracellular Vesicles for Nanoscale Flow Cytometry. All authors Morales-Kastresana A, Telford B, Musich TA, McKinnon K, Clayborne C, Braig Z, Rosner A, Demberg T, Watson DC, Karpova TS, Freeman GJ, DeKruyff RH, Pavlakis GN, Terabe M, Robert-Guroff M, Berzofsky JA, Jones JC Journal Sci Rep Abstract Extracellular vesicles (EVs), including exosomes and microvesicles, are 30-800 nm vesicles that ar (show more...)Extracellular vesicles (EVs), including exosomes and microvesicles, are 30-800 nm vesicles that are released by most cell types, as biological packages for intercellular communication. Their importance in cancer and inflammation makes EVs and their cargo promising biomarkers of disease and cell-free therapeutic agents. Emerging high-resolution cytometric methods have created a pressing need for efficient fluorescent labeling procedures to visualize and detect EVs. Suitable labels must be bright enough for one EV to be detected without the generation of label-associated artifacts. To identify a strategy that robustly labels individual EVs, we used nanoFACS, a high-resolution flow cytometric method that utilizes light scattering and fluorescence parameters along with sample enumeration, to evaluate various labels. Specifically, we compared lipid-, protein-, and RNA-based staining methods and developed a robust EV staining strategy, with the amine-reactive fluorescent label, 5-(and-6)-Carboxyfluorescein Diacetate Succinimidyl Ester, and size exclusion chromatography to remove unconjugated label. By combining nanoFACS measurements of light scattering and fluorescence, we evaluated the sensitivity and specificity of EV labeling assays in a manner that has not been described for other EV detection methods. Efficient characterization of EVs by nanoFACS paves the way towards further study of EVs and their roles in health and disease. (hide) EV-METRIC 14% (43rd 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 (d)(U)C Adj. k-factor 156.9 (pelleting) / 41.45 (washing) Protein markers EV: None non-EV: None Proteomics no Show all info Study aim New methodological development, Technical analysis comparing/optimizing EV-related methods Sample Species Mus musculus Sample Type Cell culture supernatant EV-producing cells DC2.4 EV-harvesting Medium EV-depleted serum Preparation of EDS overnight (16h) at >=100,000g Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 70 Pelleting: rotor type Type 70 Ti Pelleting: speed (g) 100000 Pelleting: adjusted k-factor 156.9 Wash: time (min) 70 Wash: Rotor Type TLA-120.1 Wash: speed (g) 100000 Wash: adjusted k-factor 41.45 Characterization: Protein analysis None Protein Concentration Method BCA Protein Yield (µg) 1.186 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 80-180 EV concentration Yes Particle yield 4.8E+11 particles/ml start sample Particle analysis: flow cytometry Flow cytometer type Beckman Astrios EQ Hardware adjustment NanoFACS system Calibration bead size 0.1;0.2;0.5

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EV-TRACK ID	EV160003
species	Mus musculus
sample type	Cell culture
cell type	DC2.4
condition	Control condition
separation protocol	(d)(U)C
Exp. nr.	1
EV-METRIC %	14