ES Cell Culture without PMEF Feeder Cells

Thaw a vial containing 1x107 ES cells into 4 mL of ES Cell Medium (containing ESGRO supplement at 1000 units/mL) and 4 mL of FBS. Centrifuge (3–5 minutes) and resuspend the cells in 10 mL of ES Cell Medium. Plate the ES cells onto the gelatinized plates at a density of 1–1.5 x 106 cells/ 25 cm2 (~3 x 106 cells/100 mm plate). Incubate the plates at 37°C with 5% CO2. Cell images are provided in this protocol for reference.
Examine the cells daily to determine if a change of media is required (indicated by a change of media color to yellow). After 2–3 days, ES cell cultures will become crowded with large colonies. At this point, passage ES cells at a 1:2 ratio.
To passage ES cells, prepare two 100 mm gelatinized plates in advance as described. Remove ES Cell Medium, wash plates twice with DPBS, and add 1.2 mL of Trypsin. Incubate plates at 37°C for 2 minutes, and then add 10 mL of ES cell medium. Pipette vigorously to break up
Add 5 mL of the cell suspension to each gelatinized plate containing 5 mL of ES Cell Medium. Excess ES cells can be frozen at a concentration of 2–10 x 106 cells per vial for future use. Please note that ES cells should always be passaged the day before you intend to electroporate.

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