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Protocols: ES Cell Culture - Gene Targeting - Protocols

FROZEN  FEEDERS


THAW OUT  CELLS IN THE NORMAL WAY, AFTER CENTRIFUGATION ,  ASPIRATE  
OFF THE SUPERNATANT AND RESUSPEND CELL PELLET IN  120 MLS OF MEDIA.   
THIS SHOULD GIVE YOU A DENSITY  OF:   3.5 X 10 5 CELLS/ML, BECAUSE  
THE TOTAL NUMBER OF CELLS  =  4.2 X 10 7 CELLS.     PLATE OUT THE 
CELLS ON THE  10CM PLATES, 12 MLS/PLATE AND SWIRL  PLATES.    
REMEMBER TO USE PLATES THAT HAVE BEEN PRE-TREATED WITH GELATIN  
(2 HOURS PRIOR THIS).

1 VIAL = 1 ML =   4.2  X 107  CELLS  =  10 X 10 CM PLATES

10  X 10CM PLATES  =  10 X 6-WELLS PLATES  =  10 X  24  
WELL PLATES  =  30 X 6 CM

1 X 10CM PLATE  =  12 MLS  =  1 X 6-WELLS PLATE  =  
1 X 24 WELL PLATE  =   3 X 6 CM

The 96-wells plates and 48-wells plates require a dilution, because 
of different cell density.

Feeders/STO Media:   DMEM;  7%  FCS,  1 X GPS