I want to convert Thp-1 monocytes to M2 macrophage. For that I'm using PMA for converting Thp-1 to macrophage then I want to activate these macrophages to M2 macrophages. For that I want to use TGF beta. Is it possible.
why not to use m-csf. I feel that would be great. But be careful of the mechanistic cross talk of PKC pathway induced by PMA and the effect of MCSF.
I am not sure if that is a good model, the PMA activation can have a wide array of effects
If I were you, I would use a combination of TGFbeta and IL-4. Combining those two cytokines, you may have a better polarization. I would not recommend using PMA, as PMA usually induce no polarization towards M2 phenotype.
Kind regards,
Raphael
Dear colleagues, I just want to point your attention to the problem that PMA impairs both alternative activation as well as deactivation of THP-1 macrophages (see references below). As incubation with PMA for at least 72 to 96 hour is vital to mature THP-1 suspension cells into adherent macrophages this is a serious drawback of the model. However, we have been recently faced with this problem while using a transfection procedure that has been developed and published by my Group, particularly for the transfection of THP-1 as well as primary macrophages. To address this problem, we have developed a protocol using PMA concentrations lower than that recommend as the standard protocol (see below). I cannot recommend a fixed concentration of PMA to avaid this problem as this depends on the Serum and PMA used for maturing the cells. Based on my own experience, I would recommend to mature the THP-1 cells with something between 10 and 20 ng/ml PMA for 72 h to 96 h (the latter time period may cause significant detachment of the cells with 10 ng/ml). Please note that this requires some kind of optimization of your model. Depending on the following experimental design I would recommend to incubate the mature cells with either 2.5 ng/ml or less PMA depending on the time period of incubation. Do not hesitate to contact me if you have further questions.
Maeß MB, Wittig B, Lorkowski S. Highly Efficient Transfection of Human THP-1 Macrophages by Nucleofection. J Vis Exp 2014; (91). doi: 10.3791/51960.
Maeß MB, Wittig B, Cignarella A, Lorkowski S. Reduced PMA enhances the responsiveness of transfected THP-1 macrophages to polarizing stimuli. J Immunol Methods 2014; 402(1-2):76-81. doi: 10.1016/j.jim.2013.11.006.
Park EK, Jung HS, Yang HI, Yoo MC, Kim C, Kim KS. Optimized THP-1 differentiation is required for the detection of responses to weak Stimuli. Inflamm Res 2007; 56(1):45-50.
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