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