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Sunday, Day 5
Thaw ES cells as follows: Suction the existing MEF media off one of the inactivated MEF 10 cm dishes. Refeed the dish with 12 ml ES media. Thaw one frozen vial of RW4 cells (1x106 cells) in a 37oC waterbath. When the last bit of ice has melted, spray the tube with 70% ethanol and transfer the contents of the vial to the inactivated MEF dish. Rock the dish to evenly disperse the cells. Incubate overnight at 37oC, 5% CO2, and 86% humidity. The T-150 flask of ongoing MEFs is ready to be expanded today. Rinse with PBS, add 7ml Trypsin/EDTA, for 10 minutes, then add 10 ml of MEF media, and pipette up and down several times. Pipette 8 ml of these cells into a new T-150 flask containing 25 mls of MEF media. Refeed the existing T-150 with 25 ml MEF media to create 2 T-150's of expanding MEFs.
Monday, Day 6
Feed the ES cells with 12 ml ES media. Approximately 60% of the ES cells will form colonies in the dish. They are football shaped, shiny, and plump. You will need 4 x 10 cm dishes of irradiated MEFs for your electroporation on Tuesday. You have 2 T-150's ongoing from which to make these 4 dishes. Follow the procedure for mitotically inactivating MEFs on Day 4. However, use both T-150s this time. Don't forget to refeed them, or you will not have MEFs for the end of the week. If the 2 T-150 flasks of MEF cells are not confluent today, feed them, and then inactivate them on Tuesday morning. If you inactivate your MEFs on Tuesday, you must either give them 2 to 3 hours to attach before changing the MEF media to ES media or initially inactivate and plate them in ES media.
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