Sean Lauber:Freezing Mammalian Cells

I like to freeze down enough cells to comfortably expand them in a T25. Depending on the cell line this number could vary. I always 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).

1. Prepare cells for collection (trypsinize, scrape, etc.)
2. Collect cells into an appropriate vessel (50 ml falcon tube)
3. Spin at 800 RPM/4*C/5 min to pellet the cells
4. Remove the supernatant and resuspend in appropriate media supplemented with 20% FBS/5% DMSO
5. Remove an aliquot of cells for viability counting if desired
6. Pipette 1 ml of the cell suspension into labeled cryovials
7. Store at -80*C overnight in a Mr. Freezie container (containing isopropanol)
8. The next day remove the cryovials to liquid nitrogen