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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. From the Laboratory of Dr. Allan Bradley Baylor College of Medicine, Houston, Texas