I am isolating murine macrophages from the bone marrow (BMDM). I was able to isolate them and grow them in culture using m-CSF. They grow nicely and I have checked by flow cytometry that about the 98% of the cells are CD11b+/CD11c-. I extracted from a WT and KO mice and I would like to characterize the macrophages for an specific genetic profile before the polarization of them using IL4, LPS, etc... Also I would like to know what kind of functional assays could be performed within this kind of undifferentiated stage. Thanks.
Dear Francisco
The best way is to pulldown your sequences. How different are the macrophages matching the genetic profiles?
Best
Prash
This is something that I don't know yet, no one has analyzed this macrophages from the WT/KO mice. I would like to do it by qPCR because I have all the tools and primers.
I see, that would certainly be a good option lest you have true positives.
I suggest you read the following paper- Immunity. 2014 Jul 17;41(1):14-20. doi: 10.1016/j.immuni.2014.06.008
In it the authors describe macrophages grown in M-CSF/CSF-1 as being baseline macrophages. The authors state that the term "activated" rather than"polarised" should be used when discussing cytokine-induced changes to their expression profiles and behaviour. However, macrophages grown in CSF-1 are well differentiated already in that they are adherent, migratory and CSF-1 dependent so they can be tested in all sorts of functional assays.
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