Splitting cells without centrifugation - (Jul/14/2016 )
Hi all. Coming back to cell culture after a good few years so apologies for the simple question, but a bit rusty with it right now. Normally when splitting cells in the old lab, say if I wanted to do a 1:5 split on an adherent cell line, would trypsinise, neutralise with media, centrifuge and then resuspend pellet in 5 ml media. 1 ml would then go into a new flask.
My new lab doesn't centrifuge cells so was a bit confused on how splitting cells would work. Normally they trypsinise (approx with 3 ml) and then neutralise with an equal volume of media. From this cells would be split into a new flask. How would say a 1:2 dilution work then if cells are to be neutralised with an equal volume of media? E.g. 3 ml trypsin + 3ml media = 6ml - then 1 ml into a new flask would be a 1:6 split?! Thanks!
You are correct - all you have to think about is the total volume containing the cells and how much of that you are using to seed the new flask. The initial volume is irrelevant as it is the cell concentration (cells per ml) you are interested in.
bob1 on Thu Jul 14 16:28:55 2016 said:
You are correct - all you have to think about is the total volume containing the cells and how much of that you are using to seed the new flask. The initial volume is irrelevant as it is the cell concentration (cells per ml) you are interested in.
Thanks very much for the reply. Would it be possible if you could give an example please just so I could get my head round it. For example after adding 3ml trypsin (no centrifugation), how would I go about doing say a 1:2 or a 1:3 split? E.g. with 20 ml media to go into the new flask. Thanks!
Sure:
Add 3 ml trypsin, neutralize with a further 3 ml complete medium... 6 ml total. For a 1:2, you would take 3 ml of this into a new flask and add 20 ml of medium. For a 1:3 2ml, into the new flask.
bob1 on Thu Jul 14 22:00:49 2016 said:
Sure:
Add 3 ml trypsin, neutralize with a further 3 ml complete medium... 6 ml total. For a 1:2, you would take 3 ml of this into a new flask and add 20 ml of medium. For a 1:3 2ml, into the new flask.
Thank you very much, this has been a great help!