Liquid differentiation of ES cells-10 cm plate
- 1 ml 1x trypsin, RT 2min.
Want cells to just start comming off as flakes from the monolayer. Check in microscope.
- Add 5ml of ES media to each plate to stop trypsin. Pool.
- Spin 1500 rpm, 5min.
- 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.
- Split each confluent plate (10cm) into 3 plates each (10cm).
- Add 9ml of differentiation medium to each bacterial plate.
- Add 1ml cells.
- On alternating days, remove 1/2 of media and add new media.
- Cystic embryoid bodies should start beating around day 10-14 after initiation of differentiation.