Hello,

I have been trying to polarize PBMC derived monocytes into M1 macrophages, using varying concentration of LPS and IFNg in combination, however upon every attempts despite using pro-inflammatory induction (LPS and IFNg), the FACS results shows macrophages expressing CD206 (M2 marker) higher compared to CD86 (M1 marker). Upon obtaining the pellet of PBMC layer, the cell pellet are resuspended with SFM and seeded into wells for cell attachment for about 3 hours. Upon cell attachment, the SFM are discarded and M-CSF medium (50ng/ml) is added into the wells (for macrophage differentiation) and left for 5 days without medium change. On the 6th day, the medium is discarded and a medium comprising 50ng/ml of MSCF, 50ng/ml of IFNg and 10ng/ml of LPS is added into the well and left for 48 hours. After that, the cells are detached and stained with CD14, CD206 and CD80 together as mixed stain and later, analyzed with FACS. The MFI value for CD206 shows to be higher in comparison to CD80 MFI value. I have also used varying concentration combination of LPS and IFNg, using the same incubation period as mentioned above, yet encounter the same issue at every attempt (CD206 is highly expressed compared to CD80). In all the attempts, M-CSF concentration was fixed to 50ng/ml. I would like to know which point is misleading.

Thank you for your help in advance.