MITOMYCIN C TREATMENT OF PMEFs

Cultures to be treated should be sub confluent ie actively growing.

1. Add 1/20 volume Mitomycin C (200 ug/ml 10 ug/ml), to culture and incubate at 37� for 2hrs.

2. After approx. 2 hrs remove medium and collect into a waste bottle. Cytotoxic waste needs to be kept separate for incineration.

3. Wash monolayer with 3 x 10 ml PBS (80 cm2 flask), 4 x 10 ml (175 cm2 flask), collect PBS into waste bottle.

4. Add TRYPSIN/EDTA (1 ml 80 cm2 flask, 2 ml 175 cm2 flask) and place flask on 37� warning tray until cells begin to come off.

5. Return to hood, hit flask on the end to dislodge cells. Add an equal volume of medium, transfer cells to a 10 ml tube. Take a sample to count. Seed cells at appropriate concentration (see chart).

6. Feeder plates can be used 2 hrs after seeding, and for up to one week later.

MITOMYCIN C SIGMA 0503 (2 mg/ampoule)

Add 5 ml DMEM (no FCS) to ampoule, transfer to a 10 ml tube, rinse ampoule with a further 5 ml (total 10 ml) Conc = 0.2 mgs/ml = 200 ug/ ml (= 20 x). Filter sterilize.

Use at 1/20 to give a working concentration of 10 ug/ml.

Store at 4� wrapped in foil as mitomycin C is light sensitive. Don't use after 6 weeks old.

NB. Mitomycin C is very cytotoxic, take care not to spill any on skin. Wear gloves and always handle in hood. See Sigma hazard sheet for further details.