Dear Richard Mate ,

You might want to try to stop the proliferation of your fibroblasts before establishing the coculture. I have used Mitomycin C (DNA cross linker) to stop the proliferation of vascular smooth muscle cells (in single culture), before coculturing them with macrophages over weeks.

kind regards

Soenke

Dear Sönke Weinert,

Thank you very much for your suggestion — it’s a really good idea. My question would be now maybe you have some experience with it, whether this treatment could potentially cause further damage to the fibroblasts over a longer period (2–3 weeks)? In any case, I will definitely give it a try.

With thanks Richard

Dear Richard Mate ,

of cause there will be a slight loss of fibroblasts. Since Mitomycin C is a non selective DNA cross linker it might happend, that you are lossing a few cells, in which (at least in my explaination with no scientific proof) important genes might have been cross linked and thereby inactivated and subsequently the cells are dying. What I also observed that some cells might not be hit by the Mitomycin C and thereby some islands of proliferation might be observable.

You should start with different cell densities, concentrations and incubation times. I was using 2 µg/ml final concentration for 1 h within the VSMC media. You should be aware after solving and aliquoting the Mitomycin C I stored it at -20°C. That will lead into non resolved Mitomycin C after thawing. (visibile by brow/black debree within the aliquot (which was pale light gray at the beginning). You can use an ultra sonic bath to resolve the Mitomycin C again in the aliquot. I never used used and refrozen aliquotes more than three times (to avioid degradation by repeated thaw and freeze cycles).

best wishes

Soenke