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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV180007	1/1	Bos taurus	Embryo culture	DG UF	Pavani KC	2018	100%
Study summary Full title Isolation and Characterization of Functionally Active Extracellular Vesicles from Culture Medium Conditioned by Bovine Embryos In Vitro. All authors Pavani KC, Hendrix A, Van Den Broeck W, Couck L, Szymanska K, Lin X, De Koster J, Van Soom A, Leemans B Journal Int J Mol Sci Abstract Extracellular vesicles (EVs) play a possible role in cell⁻cell communication and are found in vari (show more...)Extracellular vesicles (EVs) play a possible role in cell⁻cell communication and are found in various body fluids and cell conditioned culture media. The aim of this study was to isolate and characterize EVs in culture medium conditioned by bovine embryos in group and to verify if these EVs are functionally active. Initially, ultracentrifuged bovine serum albumin (BSA) containing medium was selected as suitable EV-free embryo culture medium. Next, EVs were isolated from embryo conditioned culture medium by OptiPrep^TM density gradient ultracentrifugation. Isolated EVs were characterized by nanoparticle tracking analysis, western blotting, transmission, and immunoelectron microscopy. Bovine embryo-derived EVs were sizing between 25⁻230 nm with an average concentration of 236.5 ± 1.27 × 10⁸ particles/mL. Moreover, PKH67 EV pre-labeling showed that embryo-secreted EVs were uptaken by zona-intact bovine embryos. Since BSA did not appear to be a contaminating EV source in culture medium, EV functionality was tested in BSA containing medium. Individual embryo culture in BSA medium enriched with EVs derived from conditioned embryo culture medium showed significantly higher blastocyst rates at day 7 and 8 together with a significantly lower apoptotic cell ratio. In conclusion, our study shows that EVs play an important role in inter embryo communication during bovine embryo culture in group. (hide) EV-METRIC 100% (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 Embryo culture 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 UF Protein markers EV: TSG101/ CD63/ CD9 non-EV: ApoA1/ Argonaute-2 Proteomics no Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Bos taurus Sample Type Embryo culture Separation Method Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5% Highest density fraction 40% Sample volume (mL) 1 Orientation Top-down (sample migrates downwards) 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: speed (g) 100000 Ultra filtration Cut-off size (kDa) 10 Membrane type Regenerated cellulose Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Detected EV-associated proteins CD9, CD63, TSG101 Not detected contaminants Argonaute-2, ApoA1 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/ Immune-EM EM protein CD63 Image type Close-up, Wide-field

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EV-TRACK ID	EV180007
species	Bos taurus
sample type	Embryo culture
condition	Control condition
separation protocol	DG UF
Exp. nr.	1
EV-METRIC %	100