I'm trying to polarize human monocytes to M1 or M2 phenotypes. I cultured them for 5 days in RPMI medium +10% FCS with 50 ng/ml GM-CSF (for M1) and RPMI medium +10% FCS with 50 ng/ml M-CSF (for M2), and cultured them for 2 days more after adding TNFa and IFNg - 1ng/ml and 5ng/ml respectively (for M1), and IL-10 (20ng/ml) for M2. After doing this, I found that M1 positive for CD209, CD163, and CD206 which are markers for M2? Also, I found M2 positive for CD38, CD80, and CD120b which are markers for M1??
Could you please advise me what I did wrong or what should I do solve this issue
For the M1, I don't think you are adding enough IFNg. I've seen much higher concentrations (try 50ng/mL)
For M2... why are you using IL-10? Are you pushing for M2c? IL-4 usually works better. I thought for IL-10 you need to also add TGF-b1?
Hi Mohammad Alkhrayef,
Maybe you can try to re-stimulate the monocytes (with GM-CSF for M1 and M-CSF for M2) by adding the same concentration of growth factors in the third day of culture without changing medium.
I hope it works
You can have a look this article for human M1 and M2 markers
Article Phenotypic, Functional and Plasticity Features of Classical ...
Hi there Mohammad,
The 5 day culture you are doing should be fine and the 2 day activation as well; however, I have seen better results growing cells for 6 days (replenishing media on day 3) and 24 hours of activation. For M1 macrophages, they are classically activated by LPS ( I recommend 50ng/ml) and IFNg (100ng/ml), and for M2 IL-4 and IL-13 (20ng/ml) work just fine. Try those simulations and you should have better results in your experiments. Also, remember to be gentle with the monocytes when you are isolating them, to not activate them to much; using a DBPS buffer ( without calcium and magnesium and supplemented with 2% FBS keep the monos happier) Good luck.
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