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Cell Thawing

1. Aliquots (vials are thawed rapidly and reseeded within recommended sowing range.

*When thawing, most cell death occurs between –50° C and 0° C.

2. Always check cells the following day for healthy, viable cells and freedom from contamination.

Protocol for 1x10⁶ cells/vial:

1. Remove vial from liquid nitrogen and immerse immediately into 37° C water bath until the medium is liquid, (approximately 2 minutes), and constantly agitate.
2. Tighten vial cap, ethanol vial thoroughly on outside. Add 0.5 ml of medium into the vial, drop by drop. Add cell suspension into 8 ml of fresh medium in a 15 ml conical tube (9 ml total).
3. Plate cells in two 10 cm dishes, 3 ml and 6 ml, respectively.
4. Place in the incubator, and once the cells have attached, renew with fresh medium (do this no later than 24 hours). Observe cells daily for growth and freedom from contamination.