Cell Thawing/Cell Freezing Protocol
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Freezing Cells :
- Cells should be growing well or known to be in log phase
- Count, collect and pellet cells in a 15mL test tube
- Resuspend in freezing media so that the concentration is no more than 5x10^6 cells/mL of cold freezing media
- Transfer 1mL of cells to appropriately labelled cryovials and maintain on ice for approximately 30minutes
- Transfer vials to -80C freezer for 24hrs
- Transfer to liquid nitrogen dewar or -140C freezer for long-term storage.
- Freezing media
- 10% DMSO
- 90% FCS
- you'll need 1mL per 5x10^6 cells
Thawing Cells :
- Remove vial from Liquid Nitrogen or -140C freezer and immediately transfer to 37C water bath
- While holding the tip of the vial, gently agitate the vial, being careful hnot to allow water to penetrate the cap or seal
- When completely thawed, transfer contents of vial to 15mL test tube
- Slowly add 10mL warm complete media and spin at 1000g for 5min
- Decant media and resuspend pellet in a volume of complete media appropriate for flask or macrowell
- Transfer cells to flask or 24 well plate and incubate at 37C and 5%CO2
- Cells can be checked visually or counted, beginning at approximately 1hr, for an estimate of viability. Immediate cell counts can be misleading
