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.  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 pipeting up and down several times. 
3. Centrifuge for 10 min at 400 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.