- Grow MEFs until they are 90% confluent
- Reconstitute Mitomycin C (2 mg per 200 ml MEF medium) and use at 16 ml/ T175 flask
- Incubate cells in Mitomycin C solution for at least 3 hours
- Coat plates/wells with 0.1% sterile gelatin for at least 30 minutes
- Rinse flasks liberally 3 to 4 times with 20-25 ml of PBS to remove all traces of Mitomycin C
- Trypsinize MEFs and harvest
- Count MEFs using a hemocytometer
- Plate MEFs at a low density (approximately 187,500 cells per well in a 6 well plate)
- Place at 37 oC until ready for use (keep plates up to 2 weeks, changing the medium every 3rd day)
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