Note: Mitomycin C is a cytotoxic antitumor agent and must be handled carefully; it works by cross-linking the DNA, which blocks cell division. Follow your institution's rules for safe handling and disposal. Handlers should wear latex or nitrile protective gloves and work in a biological safety or fume hood. One effective method is to inactivate the mitomycin C with an equal volume of household bleach. Inactivation is rapid.
1. Remove the feeder cell medium.
2. Add 10 mL/75 cm2 of mitomycin C medium.
Note : Make sure the entire flask is covered with mitomycin C medium so that the inac-tivation is complete and all cells are exposed for the entire incubation time.
4. Remove mitomycin C solution and inactivate it with bleach or other recommended procedure.
5. Wash inactivated feeder layer three times with 10 mL each of PBS.
6. Trypsinize the cells to remove from flask, resuspend in feeder cell medium, and re-plate the cells on the appropriate configuration of gelatin-coated (see below) culture dishes to meet experimental goals, and incubate overnight.
Note: at this point, inactivated feeder cells can be cryopreserved for later use. Be sure to indicate on the freezing vial that the cells are already inactivated.
7. The next morning, wash the dishes of inactivated fibroblasts with PBS and re-feed with either feeder cell medium or hESC medium in preparation for hESC culture.
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