Tuesday, June 29, 2004
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. Recipes Supplies Tips (责任编辑:泉水) |