Working Cell Bank
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.
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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.
