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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV190022	1/1	Bos taurus	Bovine embryo culture media	DG (d)(U)C SEC	Krishna Chaitanya Pavani	2020	78%
Study summary Full title The Separation and Characterization of Extracellular Vesicles from Medium Conditioned by Bovine Embryos All authors Krishna Chaitanya Pavani, Xiaoyuan Lin, Joachim Hamacher, Wim Van Den Broeck, Liesbeth Couck, Luc Peelman, An Hendrix and Ann Van Soom Journal Int J Mol Sci Abstract Extracellular vesicles (EVs) have been identified as one of the communication mechanisms amongst emb (show more...)Extracellular vesicles (EVs) have been identified as one of the communication mechanisms amongst embryos. They are secreted into the embryo culture medium and, as such, represent a source of novel biomarkers for identifying the quality of cells and embryos. However, only small amounts of embryo-conditioned medium are available, which represents a challenge for EV enrichment. Our aim is to assess the suitability of different EV separation methods to retrieve EVs with high specificity and sufficient efficiency. Bovine embryo-conditioned medium was subjected to differential ultracentrifugation (DU), OptiPrepTM density gradient (ODG) centrifugation, and size exclusion chromatography. Separated EVs were characterized by complementary characterization methods, including Western blot, electron microscopy, and nanoparticle tracking analysis, to assess the efficiency and specificity. OptiPrepTM density gradient centrifugation outperformed DU and SEC in terms of specificity by substantial removal of contaminating proteins such as ribonucleoprotein complexes (Argonaute-2 (AGO-2)) and lipoproteins (ApoA-I) from bovine embryo-derived EVs (density: 1.02–1.04, 1.20–1.23 g/mL, respectively). In conclusion, ODG centrifugation is the preferred method for identifying EV-enriched components and for improving our understanding of EV function in embryo quality and development. (hide) EV-METRIC 78% (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 Bovine embryo culture media 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 SEC Protein markers EV: CD63 non-EV: Argonaute2/ APOA1 Proteomics no EV density (g/ml) 1.1 Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Bos taurus Sample Type Bovine embryo culture media Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 180 Pelleting: rotor type SW 55 Ti Pelleting: speed (g) 100000 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 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 Size-exclusion chromatography Used for validation? Yes Total column volume (mL) 10 Sample volume/column (mL) 1 Resin type Sepharose CL-2B Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Detected EV-associated proteins CD63 Detected contaminants APOA1/ Argonaute2 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 133.8+-6.8 EV concentration Yes EM EM-type Transmission-EM Image type Close-up Report size (nm) 25-250

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EV-TRACK ID	EV190022
species	Bos taurus
sample type	Bovine embryo culture media
condition	Control condition
separation protocol	DG (d)(U)C SEC
Exp. nr.	1
EV-METRIC %	78