Reagents / Solutions

• Frozen cell line from liquid N₂ tank..
• Appropriate growth media (e.g., DMEM/10% FCS)
• Fetal calf serum (FCS).

Protocol  (6/1/00)

1. Make 20ml growth media/10% FCS for each vial of cells to be thawed.
2. Remove vial(s) of cells and thaw rapidly by hand suspension in the 37 degree water bath.
3. Once thawed, add media/10% FCS  to the vial very slowly.  First ml should take ~ 1 min, second ml should take ~ 45 seconds etc. Triturate cells and then transfer to a 10 ml conical centrifuge tube.
4. When cells are resuspended in 10ml spin down in bench-top centrifuge and resuspend in 10 ml media/10% FCS to get rid of remaining DMSO (note that this step has been omitted by some labs).
5. Place cells in a T25 flask and allow to settle
6. Allow to grow overnight.
7. Assess cell number and viability, if the cell density is too high for the normal growth of the cell line split cells into multiple flasks.

Dr. Herbert Geller, Head
Developmental Neurobiology, NHLBI, NIH
Building 10, Room 8C-213, 10 Center Drive, MSC-1650
Bethesda, MD 20892-1650
Email: gellerh@nhlbi.nih.gov

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