General procedure for passing (splitting) cells - (Apr/09/2008 )
Could anyone tell me how they typically passage cells?
I aspirate my media and then wash the cells with HBSS, then trypsinize them. Then I add media, spin them down and get rid of the media. I then resuspend the pellet with new media and place in a new flask.
Is spinning necessary since adding media (with serum) would inhibit the trypsin? Could I just add new media to the trypsinized cells and place however much that I need of this into a new flask?
It is not always necessary to spin, you can just add media to the flask and then add some into the new flask. If you add 2ml of trypsin, for example, you can add 8ml of media and then add 1ml of that too a new flask (1:10 dilution).
I usually do 2 washes with sterile PBS before adding the trypsin and then don't spin for most of my cell lines.
I aspirate my media and then wash the cells with HBSS, then trypsinize them. Then I add media, spin them down and get rid of the media. I then resuspend the pellet with new media and place in a new flask.
Is spinning necessary since adding media (with serum) would inhibit the trypsin? Could I just add new media to the trypsinized cells and place however much that I need of this into a new flask?
I would also probably change the media once the cells sit down. The serum can take care of the trypsin but most trypsin that people use also has EDTA in it so changing the media would help to get rid of that.
I aspirate my media and then wash the cells with HBSS, then trypsinize them. Then I add media, spin them down and get rid of the media. I then resuspend the pellet with new media and place in a new flask.
Is spinning necessary since adding media (with serum) would inhibit the trypsin? Could I just add new media to the trypsinized cells and place however much that I need of this into a new flask?
I would also probably change the media once the cells sit down. The serum can take care of the trypsin but most trypsin that people use also has EDTA in it so changing the media would help to get rid of that.
I totally agree- if you don't want to spin, then you should change the media once the cells have settled down. Otherwise, having the spin would help get rid of any residual EDTA from the Trypsin.