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Experiment number
  • If needed, multiple experiments were identified in a single publication based on differing sample types, isolation protocols and/or vesicle types of interest.
Species
  • Species of origin of the EVs.
Isolation protocol
  • Gives a short, non-chronological overview of the different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
Details EV-TRACK ID Experiment nr. Species Sample type Isolation protocol First author Year EV-METRIC
EV180007 1/1 Bos taurus Embryo culture DG
Ultrafiltration
Pavani KC 2018 100%

Study summary

Full title
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 OptiPrepTM 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
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation 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
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
DG + Ultrafiltration
Protein markers
EV: CD9/ CD63/ TSG101
non-EV: Argonaute-2/ ApoA1
Proteomics
no
Show all info
Study aim
Technical analysis comparing/optimizing EV-related methods
Sample
Species
Bos taurus
Sample Type
Embryo culture
Sample Condition
Control condition
Isolation Method
Density gradient
Density medium
Iodixanol
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
Antibody details provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD9, CD63, TSG101
Not detected contaminants
Argonaute-2, ApoA1
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
133.8±6.8
EV concentration
Yes
EM
EM-type
Transmission-EM/ Immune-EM
Image type
Close-up, Wide-field
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