Compared with 4T1-M CM, 4T1-N2 CM inhibited TAM differentiation and activation in PEMs. (A) To confirm the purity of the obtained PEMs, the cells were stained with anti-F4/80. The purity of PEMs was analyzed by flow cytometry. (B) PEMs were seeded in a 6-well plate (8 × 105) and treated with 4T1-M CM or 4T1-N2 CM for 48 h. Cell morphology images were captured using a microscope (×20). (C) The cells were stained with specific antibodies aganist CD11b, F4/80, CD206, PD-1 and PD-L1 for cell analysis. The expression levels of TAM differentiation marker (CD206) and TAM activation markers (PD-L1 and PD-1) were measured by flow cytometry, and these data were analyzed using FlowJo software. (D-H) The mRNA expression levels of TAM differentiation markers (MR1, YM1 and FIZZ1) and activation markers (IL-10 and iNOS) were measured by quantitative real-time PCR. The results are presented as the mean ± SEM. The data are representative of at least three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
