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RESEARCH DIVISION Laboratory Manual

 
 

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°C 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°C 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°C wrapped in foil as mitomycin C is light sensitive. Don't use after 6 weeks old.

Note: 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.

 

Disclaimer: The following set of protocols were contributed by various members of our lab (past and present): Christine Andrews, Fiona Christensen, Neil Della, Ross Dickins, Debbie Donald, Andrew Holloway, Gary Hime, Colin House, Yinling Hu, Rachael Parkinson, Nadia Traficante, Hannah Robertson, Ping Fu and Dennis Wang. Special thanks to Vicki Hammond, Frank Kontgen and Maria Murphy, who contributed many of the ES cell protocols. Sections dealing with Photomicroscopy, Polyclonal and Monoclonal Antibody Production were provided by members of Gerry Rubin's Laboratory (Berkeley). Any comments in the methods (technical errors etc.) E-mail: d.bowtell@pmci.unimelb.edu.au
David Bowtell PMCI October 1998