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Liquid differentiation of ES cells-10 cm plate

1. 1 ml 1x trypsin, RT 2min.
   Want cells to just start comming off as flakes from the monolayer. Check in microscope.
2. Add 5ml of ES media to each plate to stop trypsin. Pool.
3. Spin 1500 rpm, 5min.
4. Resuspend into 3 ml of differentiation medium.
   DMEM, high glucose. No Pen/Strep. No non-essential amino acids. No b-mercaptoethanol. 15% Newborn calf serum.
5. Split each confluent plate (10cm) into 3 plates each (10cm).
6. Add 9ml of differentiation medium to each bacterial plate.
7. Add 1ml cells.
8. On alternating days, remove 1/2 of media and add new media.
9. Cystic embryoid bodies should start beating around day 10-14 after initiation of differentiation.

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