Good day. Help me, please understand how to split adherent cells (Vero)....For example, I have T25 flask with cells and i need to split at the ratio 1:15 ("based on the surface area")?
you will have to detach them from the flask.
1. remove supernatant from the flask and rinse monolayer 1 - 2 times with PBS (to remove dead cells and fetal bovine serum)
2. Add 3-5ml prewarmed 0.25% Trypsin-EDTA solution to your monolayer and place back in your incubator to allow the cells to detach. should be 10-15min
3. check if detached and bang side of flask with your hand to ensure detached.
4. Add your medium (with FBS); usually an equal volume (to inactivate the trypsin)
5. Add media to new flask and then add a portion of your trypsinized cells to it.
6. EX: 5ml trypsin-EDTA (to remove cells) + 10ml media+FBS = 15ml; take 1ml of the suspension and add to new flask containing media (that is a 1:15 split).
Think of it as splitting 1 flask to 15 flasks. If you have your trypsinized cells in a total of 15ml then you would take 1ml of that suspension to a new flask (1:15).
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