Fig. 1.

Human in vitro differentiated macrophages show the characteristic M1 and M2 marker expression after polarization with respective cytokines. a Timeline shows the different treatment schedules of the naive human monocytes, differentiating and (pre‑)polarizing into the four phenotypes of the macrophages M0m‑, M0g‑, M1-, and M2-MΦ, which were used in the experiments. On day 0, PBMCs were isolated from concentrated human blood by density gradient centrifugation and monocytes were isolated by CD14+ MACS separation. On day 0, 2, and 5 the growth factors M‑CSF for M0m- and M2-MΦ and GM-CSF for M0g- and M1-MΦ were added to the medium. On day 5 the medium was renewed and the cytokines IFN‑γ and LPS for M1-MΦ and IL‑4 and IL-10 for M2-MΦ were additionally added. For the polarization analysis of the macrophages, all cells were stained with the viability stain Zombie NIR and the antibodies CD11b, CD80, HLA-DR, CD163, and CD206 (b–i). Background correction for the respective marker expression was performed by subtracting the median fluorescence intensity of the Zombie NIR and CD11b staining from the complete staining. Background corrected (ΔMFI) expression data for HLA-DR (b), CD80 (d), CD163 (f), and CD206 (h) are presented as median with interquartile range and additionally representative data for each marker (c, e, g, i) are depicted. Statistical analysis was performed with the Kruskal–Wallis test and Dunn’s correction for multiple testing comparing all four conditions with each other. Significance is indicated as: * p < 0.05, ** p < 0.01, ***p < 0.001