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 media
60 ml RPMI1640 or DMEM 40 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% DMSO 0.1 ml versene
- Filter through a 0.2 μ filter.
If using 50% DMSO: 3 ml pH adjusted medium 2 ml 50% DMSO 0.1 ml versene
- Filter through a 0.2 μ filter.
NOTE: Versene can be left out, but adding it prevents cells from clumping.
