Sean Lauber:Freezing Mammalian Cells
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When freezing cells I like to think about how many times I can split a flask into a T75 (to be ready for the next day) to give an idea of how many vials to freeze per flask. For instance, if I have a confluent T150, and I can split that T150 to make 4 T75s which would be ready for the next day (assuming a 1:2 split is appropriate for 24 hour confluency), then I would make 4 vials out of a single T150. It's a good idea to keep an additional flask with cells to ensure I still have cells even if something goes wrong with this process (and kills all my cells).
- Prepare cells for collection (trypsinize, scrape, etc.)
- Collect cells into an appropriate vessel (50 ml falcon tube)
- Spin at 1000 RPM/4*C/3 min to pellet the cells
- Remove the supernatant and resuspend in appropriate media supplemented with 20% FBS/5% DMSO. At this point try to work quickly as the DMSO is killing your cells
- Remove an aliquot of cells for viability counting if desired
- Pipette 1 ml of the cell suspension into labeled cryovials
- Store at -80*C overnight in a Mr. Freezie container (containing isopropanol)
- The next day remove the cryovials to liquid nitrogen