science noob, on 16 December 2012 - 06:38 PM, said:
Wek, on 16 December 2012 - 02:21 PM, said:
Could anyone give me brief explanation of the process in replacing a vial of cells when you plate it?
Do you grow the vial on a plate to ~80% confluency, trypsinize the cells, deactivate trypsin with medium, pellet the cells, resuspend cells in small volume (200ul) of medium, remove a small amount (~50ul) of cells for freezing and plate the remaining cells?
Also, how does passage numbering work? When exactly does it increase?
What do you mean "replacing a vial of cells"?
Yup, your procedure sounds right. You can choose to passage the cells after trypsinisation or you can freeze them down in DMSO. Some people freeze cells in DMSO and media but we use DMSO diluted in serum (10% DMSO final concentration) and slow freeze the cells.
And your question about 'passage': https://www.google.c...iw=1366&bih=635
Our lab (*and most scientists) define 1 passage or P1 as when cells leave a monolayer culture flask/plate (via trypsinisation) and plated onto a fresh new flask - this is ONE passage.
By replacing a vial I mean restocking my supply so I don't run out of vials. I was told that I should always take some cells after trypsinisation and freeze them. So after I thaw and plate a vial (at P3) I'll grow the cells to confluency, trypsinize the plate, and freeze some of it (maybe 1/3 of the cells). This new vial will become the "replacement" for the original vial I used with the same passage number. The rest of the cells will be use for experiments.
Also, when I freeze down cells, I try to remove as much media (MEMa) as I can, but there is always 1-3 microliters keeping the pellet moist. Will that affect the cells during the freezing process?
Let me see if I understand the process of passage numbering. The passage only goes up when and only when I let cells grow to confluency and need to split them to X number of plates, right? What if I freeze down the cells instead of split and plate them? Will the vials be of the same passasge or will they be a passage higher?
Edited by Wek, 16 December 2012 - 07:08 PM.