I have isolated primary mouse embryonic fibroblasts from CD-1 mice. They grow fine until P4/P5. Generally at P3, I arrest them for use as feeders. I use mitomycin C at a final concentration in the media of 10ug/ml and keep it for 2 hours. Following this, I give 2X PBS wash and 2X media wash by centrifugation. But the problem I am facing consistently is that after 12 hours of inactivation, the fibroblasts start dying off and detaching from the plate. This problem has been very consistent and I cant grow ES cells on them. The fibroblast media consists of 15%FBS, NEAA & B-ME. Please can somebody help me fix this issue?
I suggest once you remove Mitomycin and give 2 PBS washes, why don't you add HuES media on to dish & keep for 1-2 hrs followed by plating your HuES cells. :)
Hi Vishnu,
I have tried that. Actually, I am working with murine ES cells. The problem is the next day, the ES themselves peel off the culture because the underlying feeders lose viability.
A couple of suggestions. MEF media: use 20% FBS, 1% NEAA, 1%LG, 1%PS in DMEM (no BME). Also, we the MMC may be too toxic. To avoid chemRx and make things a bit easier, we just irradiate at 4500 RADS instead. The cells should have no problem. Also, are you coating your plates with 0.1% gelatin before plating down the feeders? This is important as well.
Hi Jennifer,
We don't have a gamma irradiating faciltity; thats why I have to use chemical inactivation of MEFs. I am coating my plates with gelatin. The inactivation by mitomycin C is so routine around the world I cant figure out whats going wrong. Anyways thanks for your help.
Well, may be your MMC concentration is too toxic for the cells that they do'nt survive too long. You better titrate your cells with various MMC concentrations and judge an appropriate one by observing both (1) Prolonged survival and (2) absence of feeder regrowth over about 2 weeks period after the feeders are plated alone; one should check if the untrated cells are growing normally, I mean, they are devoid of mycoplasma contamination?? The growth arrest of MEFs by MMC does not require more serum. Chemical inactivation does not necessarily differ from irradiation towards responding to growth arrest.
Thank you Lakshmana Sir. The fibroblasts normally proliferate nicely although I have not checked for mycoplasma. The concentration of MMC widely used is 10ug/ml. I was not aware that it might need titration.
Hi
Please, make sure your fibroblasts are not mycoplasma infected.
We normally use 10% FBS and wash 3x with media (DMEM+10%FBS+Glutamin + or - NEAA). Wells or flasks should be coated with gelatine before plating feeders.
If I don't missunderstand you, the final concentration of MMC in the media is 10ug/mL. Please, try to dilute 20uL of this stock MMC solution for every mL of media, and incubate the cells with MMC for 3 hours.
Moreover, I suggest you to coat the plate with 0,2% gelatine.
Hope I can help!
Hi Marta,
Pardon me but could you please explain once again? You are right about the part that the final MMC concentration in the media is 10ug/ml. Do you mean to say that I should further dilute the 10ug/ml?
Yes, once you have the working solution of MMC at a concentration of 10 ug/mL you should dilute it again using 20uL MMC/mL of media
Hello Srav,
That is exactly what I used to do because I learnt this first from Inamdar's lab.. The problems started back then and after reading a bit I found that people do the other way also of trypsinizing and giving media washes. So, just gave it a try. Thank you for your help.
Best!
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