Fig. 4

M1 to M2 polarization upon co-culturing of RAW264.7 macrophages with 4T1 tumor cells. a Flow cytometric analysis for intracellular expression of the pro-inflammatory/M1-related cytokine IL-12 and anti-inflammatory/M2-related cytokine TGF-β1 in RAW264.7 macrophages after 96 h mono-culture and co-culture with 4T1 tumor cells (n = 3 for all culture conditions). RAW264.7 macrophages derived from 4T1 + RAW264.7 co-cultures were gated (purple) based on positivity for F4/80 (green line) relative to the isotype control (blue line). The gates for IL-12 and TGF-β1 (purple) were applied for quantification of both positive signals (red line) relative to their isotype control signal (blue line). The bar graphs show the percentage of the F4/80-positive cells based on the applied gatings. b Levels of secreted pro-inflammatory/M1-related (TNF-α) and anti-inflammatory/M2-related (BAFF, G-CSF and TGF-β1) cytokines in cell-free culture media of 24 h mono- and co-cultures of RAW264.7 macrophages and 4T1 mammary tumor cells (n = 3 for all culture conditions). All data are presented as the means +/− SEM. ND: not detectable, *: P < 0.05, **: P < 0.01, ***: P < 0.001