I have been trying to culture mouse embryonic fibroblasts for quite a while now but have been facing a lot of problems with the same. I isolated the fibroblasts from E13.5 old mouse embryos (CD-1 strain). They grow fine when subcultured regularly. But, immediately after mitomycin C treating them, for using them as feeders, they start detaching from the plate after 12hours and a lot of debris appears in the medium. As a result, I cant use them as feeders. Can someone help me in identifying where the actual problem is arising? I generally inactivate them with mitomycin-C at P3 or P4.
Hi Debosree,
I work on stem cells and an easy way to get your MEFs and use them as feeders is to expose them to gamma-irradiation so they stop dividing. Here it is the link of the protocol (obtained from Current Protocols in Molecular Biology):
http://www.boneandcancer.org/MOLab%20protocols%20since%2011-2005/E20-mouse%20embryonic%20fibroblast%20%28MEFs%29%20isolation.pdf
Let me know if you can access to the PDF file, otherwise I could send it to you.
Best,
Alejandro Amador-Arjona
Hello Leonid,
The concentration is 10ug/ml in the medium and I keep for approximately 3hours. The concentration I know is standard but does the exposure time vary from strain to strain?
Hello Alejandro,
Thanks for the protocol. I could download it. Could you please suggest as to what are the sources of gamma irradiation of 4000rads? Also, for how much time should I irradiate them?
Debosree,
We use this particular gamma irradiator: Gammacell 40 Exactor
http://www.theratronics.ca/product_gamma40.html
We use a different irradiation that the one in that protocol. We use 25Gray since the machine at our facility only has that particular dose rate. Let me ask how long we exposure the cells and I will get back to you soon.
Best,
Alejandro Amador-Arjona
The concentration and time of exposure to MMC is very critical. I usually use 12ug/ml concentration in the medium for 3 hours. Another critical point is to wash the cells at least twice with PBS after treatment, presence of any residual MMC might have toxic effects on cells.
Debosree,
The 25Gray is the standard unit of absorbed ionizing-radiation dose, the conversion from Gray to Rad: 1Gray = 100Rad
Hope that helped!
Best,
Alejandro Amador-Arjona
Hi Alejandro,
Thank you so much for your response; it was very helpful. I will see if we have a gamma irradiator at our institute. Do let me know the time of exposure.
Best
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