Materials:

• Vial of frozen cells
• 37°C Water bath
• Tissue culture medium containing 5-10% serum or other protein
• Centrifuge

1. Place vial of cells in 37°C water bath and agitate until thawed.
2. As soon as the suspension is liquid, transfer it into a 15 mL centrifuge tube.
3. Slowly add 9 mL of medium containing serum. Invert tube 2 or 3 times to mix.
4. Centrifuge for 10 min at 400 x g.
5. Aspirate or decant the supernatant and gently resuspend the cell pellet in 10 mL of medium.
6. 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.