Freezing Cells

1) Keep prepared solutions on ice. 2) Determine total cell count of cells to be frozen. (e.g. 1 X 108 ) 3) Determine number of vials to be frozen. (e.g. 10 vials at 1 X 107 ) 4) Centrifuge cell suspension. 5) Resuspend cell pellet in half the required volume of freezing media. (eg if freezing 10 vials, add 5 mls freezing media) 6) Keep cell suspension on ice. 7) Slowly add dropwise with mixing an equal volume of DMSO solution. 8) Dispense 1 ml of final mixture per sterile Nunc freezing vial. 9) Freeze in liquid nitrogen plug or in controlled rate Planar freezer.

Freezing media60 ml RPMI1640 or DMEM40 ml Gammaglobulin free Horse Serum (or FCS)1 ml Penicillin - Streptomycin- Filter through 0.2µ filter.- Aliquot in 15 ml conical tubes and store in -200 C freezer for long term periods.- Aliquots can be stored in tissue culture flasks in refrigerator for short periods.20% DMSO solution MAKE FRESH ON DAY OF USE!If using 100% DMSO:4 ml pH adjusted medium RPMI (orange-red color)1 ml sterile 100% DMSO0.1 ml versene- Filter through a 0.2 µ filter.If using 50% DMSO:3 ml pH adjusted medium2 ml 50% DMSO0.1 ml versene- Filter through a 0.2 µ filter.NOTE: Versene can be left out, but adding it prevents cells from clumping.