Cell Thawing/Cell Freezing Protocol
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
