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).

  1. Prepare cells for collection (trypsinize, scrape, etc.)
  2. Collect cells into an appropriate vessel (50 ml falcon tube)
  3. Spin at 1000 RPM/4*C/3 min to pellet the cells
  4. 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
  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