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The cells should be split when the well is 70-80 % confluent (usually 2-3 days following the last split or thaw. You always want to keep the cells in log phase growth because this is when the cells are most robust and will continue to keep their toti- and pluripotent properties.
Trypsinize cells by:
1. Feed cells 2-3 hours before trypsinization
2
. Aspirate media and wash cells with PBS
3
. Add trypsin: 96-well plate - 30 ul/well
24-well plate - 100 ul/well
6-well plate - 500 ul/well
10 cm dish - 2 ml/plate
4
. Incubate for 5 min @37C
5. Add 2X ES media volume (i.e. 1 ml of ES media to .5 mls of trypsin) and pipette to single cell suspension
6. Add to 15ml conical tube and centrifuge 5 min. at rpms
7
. Discard supernatant, add ES media and pipette into single cell suspension
8
. Add ES cells to prepared feeder well/plate
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