This is a cached page for the URL ( To see the most recent version of this page, please click here.
Protocol Online is not affiliated with the authors of this page nor responsible for its content.
About Cache
Cell Thawing/Cell Freezing Protocol
The University of Chicago





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