Cell Thawing/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 (see below for freezing media recipe)
    • 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
      • 5 ml Sterile DMSO
      • 45 ml FCS
      • no more than 3x10^6 cells/1 ml/vial
      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 not 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