1. Plate wells with gelatin for 5 min. or more, remove gelatin.
2. Prepare 15 ml tubes with 3-4 mls of feeder media to dilute out DMSO
3. Thaw vial of cells quickly in 37C waterbath. As soon as ice crystal has thawed pipette contents into 15-ml tube.
4. Pipette 2-3X to dilute out freezing media
5. Spin for 5 min. at 1000 rpms
6. Remove supernatant and add enough media to plate cells at the appropriate concentration.
7. Pipette to a single cell suspension and lay down on gelatin coated plates. Feeders should be laid down at least two hours before plating ES cells.
Chart of plate size and corresponding feeder concentration
Size Feeder Concentration
10 cm dish 1X (two plates use 2X
6-well plate concentration)
24-well plate
12-well plate
96-well plate _
60 cm plate .35X
2 wells of a 6-well plate
8 wells of a 24-well plate
2 nunc 4-well plates _
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