Freezing and Thawing Cells
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Freezing Mammalian Cells
- In a T75 flask, grow cells to 80-90% confluency, or 75-80% density for suspended cells.
- Collect cells (trypsinize/scrape adherent cells, etc). Pellet using a 500g (1500RPM) spin, 5min at 4oC.
- Suspend cells in 4ml of culture media containing at least 10% FCS.
- Add 1ml of DMSO, mix well.
- Aliquot into labelled cryotubes, 0.5-1ml per tube.
- Place in a cell freezer that has equilibrated to room temperature
- Mark the container (on tape, on the lid) to indicate a use
- After the 5th use, the isopropyl alcohol must be replaced with fresh alcohol
- Put cell freezer in a -80oC freezer. This will cool the cells at ~1oC/min. Let sit in -80oC overnight.
- Transfer tubes to liquid nitrogen store
- Make sure to enter the cells into the cell storage database.
Thawing Mammalian Cells
- Warm 10ml of the appropriate tissue culture media to 37oC
- Remove a frozen tube from the liquid nitrogen store and transfer immediately to a 37oC waterbath
- Place 10ml of media into a T25 or T75 flask (T25 will achieve confluency faster). Pipette thawed cells into media
- Place in 37oC incubator. 12-18 hours later exchange the media in order to remove the DMSO