How to freeze mamalian cells
How to freeze mamalian cells
June 29, 2004
The Science Advisory Board
Description
This protocol describes how to freeze tissue culture cells (mamalian) 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.
6.) Transfer suspended cells to 50ml conical tube
7.) Pellet cells by centrifugation 1000g for 5 minutes
8.) Resuspend in media supplemented with **20%** FBS. Cells should be between 1x10^6 and 5x10^6 cells/ml.
9.) Add DMSO to get 10% DMSO.
10.) Put in 1.5 ml cryotubes and freeze at -80 degC for 3 hrs.
11.) Transfer to LN2 tank.
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