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  • Vial of frozen cells
  • 37°C Water Bath
  • Tissue Culture Medium Containing 5–10% Serum or Other Protein
  • Centrifuge

Thawing cells is simple. However, speed is key to the optimum cell recovery.


  1. Place vial of cells in 37°C water bath and agitate until thawed. It is important to thaw the cells quickly. Do NOT allow thawed cells to remain in freezing media any longer than necessary.
  2. Slowly add thawed cells to 9 mL of medium containing serum. Invert tube 2 or 3 times to mix, or mix gently by pipetting up and down several times.
  3. Centrifuge for 10 minutes at 200 x g.
  4. Aspirate or decant the supernatant and gently resuspend the cell pellet in 10 mL of medium.
  5. Remove an aliquot for cell count and proceed with experimental manipulations.


  • The cell suspension may form clumps after standing at room temperature. This can be avoided by preparing and using the cells promptly, or by adding DNase to the suspension at a final concentration of 10 units per mL.