In order to get M2 macrophages, I used THP-1 with 100 ng/ml PMA for 48h, then the cells were rested for 24h in a fresh medium without PMA. Then I put 20 ng/ml IL-4 for 48h. However, after staining the cells with CD206 for M2 and CD11b for M0, I only get about 7 % M2 and 75% M0. Some research papers and reviews recommend using PBMC monocytes instead of THP-1 because THP-1 cells are unable to be polarized to M2 and that approves the results that I get.
Have you ever used THP-1 and gotten more than 50% of M2 or TAM M2-like macrophages? Or I have to use PBMC monocytes. The problem in cell type or markers and cytokines or both? My purpose to get only more than 50% M2.
on top of adding IL-4, try adding LPS as well to differentiate THP-1 M0 cells to M2 macrophage.
After initial priming with 100 ng/ml PMA for 48h and then resting the cells in fresh medium for 48h, treat the cells with a cocktail of 20ng/ml IL4 and 20ng/ml IL13. Culture the cells for another 48h. Wash the cells and culture the cells back in fresh media fo another 48h before detaching them for staining and acquiring them on flow.
As these cells are highly adherent, detaching process will result in cell breakage and cell death. Using Trypsin may hamper cell surface markers. This will affect your results on flowcytometry. So, for detaching the cells, try incubating the cells in ice cold PBS w/o calcium and magnesium and 5%FBS on ice for 45min. Gentle scrapping will help you get cells in better condition.
Alternately, you can try using wester blotting, imaging. CD209, CD163 and TGM2 are good M2 markers to rely on. PCR is also an option to consider
Vinod Nadella You mean after 48h of IL-4, we add fresh medium without IL-4 right?
Ahmed Al-Omar did you manage to get 50% M2 macrophages?
Vinod Nadella Have you ever used Accutase for detaching the macrophages or would it hamper the surface markers too?
I never used Accutase and there is no particular reason for not using.
Accutase does have proteolytic and collagenolytic enzymes and so it may also affect cell surface markers. In fact, any enzymatic digestion solution to detach or dissociate cells will result in the cleavage of surface markers !
Ingrid Meschede Yes at least it should be 50%. actually, I need TAMs M2 like macrophages then I will do co-culture with A549.
Vinod Nadella Thank you for your reply. I asked because accutase is much lighter reagent compare to trypsin so thought it could be ok. I will try your suggestion then to use ice cold PBS w/o calcium and magnesium and 5%FBS on ice for 45min. Thanks again.
Ahmed Al-Omar Thank you for your reply. I was just wondering if your M2 polarisation protocol worked in the end?
Ingrid Meschede I am going to use PBMC with M-CSF and IL4 because THP-1 and PMA did not work with me.
Ahmed Al-Omar Thank you for sharing your experience with me. I am using primary cells too as THP1 didn't work for me either.
I use primary cells as well. As far as I know, THP-1 cell line is not an ideal model for M2 polarization. Instead, PMA-primed THP-1 is more suitable for M1 polarization. Besides, 100 ng/ml PMA may be too much as it can induce cell death based on my experience. I usually use 10-20 ng/ml PMA. With 20 ng/ml PMA, more than 90% cells will attach to the plate and start to change mophology after overnight's incubation.
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