Tuesday, June 29, 2004 Description
This protocol describes how to freeze tissue culture cells (mammalian) in a way that they will be viable when thawed. Procedure
1. Aspirate media
2. Wash cells with PBS
3. Apply trypsin/EDTA (1ml for 15cm dish)
4. Incubate 5 min in TC incubator
5. Collect in PBS (amount does not matter - just enough to get all the cells suspended)
6. Take small sample to count cells.
7. Transfer suspended cells to 50ml conical tube
8. Pellet cells by centrifugation 1000g for 5 minutes
9. Resuspend in media supplemented with 20% FBS. Cells should be between 1x10^6 and 5x10^6 cells/ml.
10. Add DMSO to get 10% DMSO.
11. Put in 1.5 ml cryotubes and freeze at -80 degC for 3 hrs.
12. Transfer to LN2 tank. Recipes
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