Reagents / Solutions

• Cells! (at least 5 x 10⁶).
• Fetal calf serum (FCS), heat inactivated at 65 °C for 1 hr.
• Dimethylsulfoxide (DMSO).
• Freezing vials - labeled with cell type, date and your initials.
• Plastic or cardboard box for initial freezing of cells
• Canes for liquid nitrogen tank
• Liquid nitrogen tank (in cold room).

Protocol (6/1/00)

1. Spin down cells, remove and discard the supernatant.
2. Resuspend the cells at 5 x 10⁶ per ml in FCS.
3. Add DMSO to 10% (by volume) dropwise, agitating gently after each drop. Do this slowly.
4. Place 1ml aliquots into the vials.
5. Put vials into a rack inside a box.
6. Place lid on box and seal in place with tape.
7. Place box in the -80º freezer. Leave for 6 - 24 hrs to freeze.
8. Remove vials from box and place in canes and lower into liquid nitrogen.  Be careful not to get splattered!
9. Test the efficiency of the freezing procedure by thawing one vial (method).
10. If the cells were healthy (> 95% viable) at the start of this procedure, then cells should be 80-90% viable 24 hours after thawing.

Dr. Herbert Geller, Head
Developmental Neurobiology Section, NHLBI, NIH
Building 10, Room 6D18, 10 Center Drive, MSC-1754
Bethesda, MD 20892-1650
Email: gellerh@nhlbi.nih.gov

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