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Isolation and characterization of mEVs and EV-depleted supernatant

LT Lingjun Tong
JW Jiong-Wei Wang
SZ Sitong Zhang

Last updated date: Oct 26, 2023 Views: 1100 Forks: 0

original An abbreviated version of this protocol was published in Science Advances
Milk-derived extracellular vesicles protect intestinal barrier integrity in the gut-liver axis
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Equipment: Beckman OPTIMA 90X, Type 70Ti Fixed-Angle Rotor

Material: chymosin (2 X Rennet Chymosin Fast-Set 7 grams Mix for 50 Litres of milk Rennin Cheese salt & Coagulant Tub, FAST-SET), calcium dichloride (CaCl2, Sigma), bovine milk collected from Holstein cows within 3 days postpartum at a local dairy farm and delivered to the laboratory immediately in a portable freezer

 

  1. Add 0.05% chymosin and 0.3% CaCl2 to bovine milk and shake thoroughly. Stand still for 30min at room temperature to solidify it.
  2. Centrifuge at 12000g, 4 degree for 30min using centrifuge to separate whey (supernatant) and curds (pellet). Use syringe to take the supernatant and go through the 0.22um filter to get rid of the rest curd and sterilize the supernatant.
  3. Send the supernatant to ultracentrifuge.

First round: 90000g (29600rpm, 1h)-get rid of floating fat and collect supernatant

Second round: 1350000g (36300rpm; 90min)-collect pellet which contains EVs

Third round: 135000g (36300 rpm; washing, 1h)-collect pellet which contains EVs

  1. After ultracentrifuge, the pellet was resuspended and the liquid was transferred onto the 100-kDa filters and centrifuged twice at 3000g for 30 min to improve mEV purity.
  2. The protein concentrations of mEVs and mEV-depleted samples were measured using the Micro BCA Protein Assay Kit (Thermo Fisher Scientific).
Copyright: © 2023 The Author(s); This is an open-access article under the CC BY-NC license (https://creativecommons.org/licenses/by-nc/4.0/).
How to cite：
Readers should cite both the Bio-protocol preprint and the original research article where this protocol was used:
  1. Tong, L, Wang, J and Zhang, S(2023). Isolation and characterization of mEVs and EV-depleted supernatant. Bio-protocol Preprint. bio-protocol.org/prep2484.
  2. Tong, L., Zhang, S., Liu, Q., Huang, C., Hao, H., Tan, M. S., Yu, X., Lou, C. K. L., Huang, R., Zhang, Z., Liu, T., Gong, P., Ng, C. H., Muthiah, M., Pastorin, G., Wacker, M. G., Chen, X., Storm, G., Lee, C. N., Zhang, L., Yi, H. and Wang, J.(2023). Milk-derived extracellular vesicles protect intestinal barrier integrity in the gut-liver axis. Science Advances 9(15). DOI: 10.1126/sciadv.ade5041

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