I want to polarize THP-1 to M0, then to M2. After adding PMA and getting macrophages, I need to get the highest M2 percentage (90%) because I can not isolate M2 from other subtypes. For this reason, Does IL-4 alone enough to polarize macrophages to M2 or I need to use other cytokines like IL-13 or IL-10? in addition, what is the most practical CD marker for M2? CD206 or CD163? there are many researchers who say that it is not easy to get CD206 so should I use CD163 instead?
Thank you
Hello Ahmed Al-Omar
You can use IL-4 and IL-13 for M2a and IL-10 for M2c.
Both CD206 and CD163 can be used as markers for M2. You can use CD163 instead as it is a highly specific marker for M2-type macrophages.
Best.
It depends on which M2 specific phenotype you are after. IL-4 and IL13 is enough to polarize macrophages to M2a. Similarly, stimulation with immune complexes lead towards M2b. Stimulation with IL10 and/or glucocorticoids leads towards M2c and with IL6 towards M2d activation !!
Both CD163 and CD206 are expressed by M2 macrophages but the expression of the receptors is differentially regulated. CD163 expression is increased in response to interleukin-10 (IL-10) stimulation, while CD206 expression is upregulated by IL-4 and IL-13. So, saying that it is difficult to see the expression of CD206 is not true.
BW
Hi all,
Thank you for you question, Ahmed Al-Omar and Malcolm Nobre and Vinod Nadella for your answers and help.
I am trying to do the same. I am following this paper where they add PMA for 24h and then add all cytokines you cited plus TGFb1 for 48h. I have two questions, is it really necessary to add TGFb1 or the other cytokines will do and second, are 48h enough for polarisation? How long do you normally incubate the cells for?
Thanks a lot in advance
BW
M2 phenotype is not only induced by IL-4 or IL-13, IL-10, immune complexes, glucocorticoids, but also by TGF-β. Depending on the type of stimuli, one can expect variation within the M2-like cells. Interleukin (IL)-4 and IL-13 are known to activate the STAT6 signaling pathway by promoting transcription ofSTAT-6-responsive genes including M2 signature genes including Arg1, Fizz1, Ym1. While TGF-β acts via SNAIL up-regulation which has been strongly implicated in the MAPK pathway. It was also shown that overexpression of SNAIL caused M2-like differentiation and promoting the expression of M2-specific markers. Both treatments stimulates the expression of CD206 of macrophages, which is a classic M2-phenotype marker. So, you can try any of your choice. They need not be combined. However, I never used TGF-β1 in my work !
For THP-1 cells, 48h stimulation is more than enough for polarization.
Vinod Nadella Thank you very much for your amazing answer and teaching me lots about macrophages. I am new to the field so I still have a long way to go. Could I ask you one more thing, if that's ok. How many THP-1 cells would plate per well in a 96well plate for polarisation? I suppose the number of cells that you plate is quite important factor for the cells to be polarised properly.
Thanks a lot again for all the help! :)
Cells at confluency per well in a 96 well plate be around 4 x 10^4. THP-1 cells once stimulated to differentiate are terminally differentiated. So, there is less chances of cell proliferation. However, to inhibit over crowding and to allow enough space for cells expecting their M2 phenotypic morphology post differentiated, I would go with 2-3 x 10^4 cells per well.
Vinod Nadella Much less than I planning to plate. Thank you so much again for all the help! You're star! :)