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1. Remove the culture media.
2. Rinse ES cells with PBS (use 1 ml PBS per 10-15mm diameter surface).
3. Add Trypsin to trypsinize ES cells. (use 0.1 ml/10-15mm diameter surface).
4. Trypsinize at 37c for 3-5 min by monitoring cell morphological changes under microscope. (stop reaction when ES cells start to detach from the surface)
5. Stop trypsinization by adding complete culture media containing FBS (use 1ml media per 10-15mm diameter surface).
6. Resuspend ES cells gently to obtain single cell suspension. Check under microscopy.
7. Spin down ES cells briefly at 200g for 3 min in a sterile tube.
8. Remove supernatant and resuspend cells in appropriate amount of ES cell culture media (1 to 6 passaging).


Reference:

Essential stem cell methods. By Robert Lanza, Irina Klimanskaya, 2009

Related books:

Stem cell methods and protocols