Science Gateway

Protocols: ES Cell Culture - Gene Targeting - Protocols

Preparing 48-Well Plate Feeders



In preparing 48-well plates, a dilution must be made from the regular
density of 3.5 x 105  cells/ml.   The required density for 48-well 
plates is:  8.2 x 104  cells/ml  (650 ml/well  or  31.2 mls/plate).

Example:

For a total of 5 x 48-well plates, the calculation is:

5 plates x 31.2 mls  =  156 mls  +  10 mls Extra  =  166 mls  (Total)

166 mls x 8.2 x 104 cells/ml  =  1.36 x 107 cells  (Total)

1.36 x 107 cells  :  3.5 x 105 cells/ml  =   38.86 mls

Dilution:  38.9 mls (of cell suspension  @ 3.5 x 105 cells/ml) + 127.1  mls Media.

Take a count at this point, to verify the cell density of 8.2 x 104 cells/ml.   
Plate-out this feeder media (dilution) into the 48-well plates.    
REMEMBER TO USE PLATES THAT HAVE BEEN GELLED, 2 HOURS PRIOR THIS.    
Dispense 650 ml of feeder media/well (31.2 mls/plate).   
Mix plates to distribute cells uniformly and incubate.


No. of Plates     Total # of Cells     Dilution (mls)     Total Vol. (mls)
 5 plates         1.36 x 107 cells     38.9  +  127.1      166  mls
10 plates         2.64 x 10 7 cells    75.0  +  247.0      322
15 plates         3.92 x 10 7 cells   112.0  +  366.0      478
20 plates         5.20 x 10 7 cells   148.5  +  485.5      634


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