I have recently started to culture RAW 264.7 cells. I have never cultured this cell line before. I noticed from a number of sources that they may also be able to differentiate into M1 and M2 subtypes in vitro, but the description was not very detailed. I do not know whether the differentiation should be done in serum free medium or just with heat-inactivated FBS. How can I check the differentiation efficiency? Is it only possible by FACS methods?
Don't use RAW 264.7 but primary macrophage. Either bone-marrow derived macrophage or peritoneal macrophage.
You can use heat inactivated FBS. You can differentiate Macs into M1 by culture in presense of IFNy and LPS or to M2 by culture in presence of Th2 cytokines IL-4 or IL-10. The easiest way to look polarization of macrophages is by real-time PCR. Moreover, using real-time PCR you can assess expression of many more M1/M2 markers.
I absolutely agrre with Johan Duchene & Samantha Moore: Do not use those cells, they are not "real" macrophages". By the way, do you work with human or murine cells? This point is important.
Thank you for the suggestions, I am planning to work on mice macrophages, I realize there are some important difference between human and mice macrophages that's why I am thinking of using raw264.7 instead of THP-1 cell lines. In that case I may just proceed to use the primary BMMs for this polarization studies.
I agree with those who posted not to use these cells but rather BM-Macs. We have been using both cell types for M1-M2 polarization and the Raw cells are NOT a good model at all.
I hope the primary cells are good for these study, M1 & M2 polarization you can look by Expression if Arg, iNOs, Il-12 / IL-10 ratio and other markers by Real time PCR
Because you wont find a clear cut population positive for only M1 or M2 markers in Vivo.and even after differentiation them. You need to check ratio of associated Markers.
As an update, RAW264.7 cells, BMDM and PM all response well to LPS (regarding the classical M1 markers like IL1b, IL6 and iNOS) but RAW264.7 cells do not response well to IL4 stimulation (we checked Arg1, Fizz1, Clec7a), while BMDM and PM response quite well... Both BMDM and PM response to IFNr but fairly week while RAW264.7 cells do not response to IFNr at all in our case... As a follow-up, just for anyone who is interested...
My Lab use 100ng LPS as postive control MI polarization. Griess assay is a 15 minute assay that test the NO production. If M1 polarization occurs, the NO production increase significantly.
Rongrong Fan Thank you so much for this update! Since IL-4 did not work for M2 polarization, what did end up working? Have you tried IL-10?