Optimally ES cells should be fed every day and split every second day (by which time they should be 70-80% confluent). It is important not to let them overgrow, since this may induce them to differentiate.
1. If the cells are split into gelatinized plate, the plates should be prepared (as detailed previously) before starting the trypsinization.
2. Trypsinize the cells by first aspirating the medium off the dishes and then rinsing twice with PBS.
3. Aspirate off any remaining PBS, and add trypsin (0.1%) to the cells. For 10-cm plates, we use 2.5 mL trypsin. This volume should be scaled according to the size of plate used.
4. Place the dish containing the cells in trypsin in a 37°C incubator for 3-6 min.
5. Check under an inverted microscope to see if the cells have detached. When they have, add 5 mL medium to the dish.
6. Resuspend the cells and transfer them to a 12-mL tube.
7. Spin down at 1000g for 5 min at room temperature.
8. Aspirate the medium, and then add 1 drop of PBS to the pellet.
9. Flick the tube hard in order to resuspend the cells.
10. Add 5 mL of medium to the tube, pipet again to mix, and split the contents at 1:5 or 1:7 ratio in new plates containing sufficient volume of medium.
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