Supplementary Figure 1: In vitro analysis of phagocyte polarization in iNOS-tdTomato-Cre and Arginase-YFP mice.
From: Mononuclear phagocytes locally specify and adapt their phenotype in a multiple sclerosis model
(a) Confocal images of tdTomato-specific immunostaining (above, red) and quantification (mean indicated as a grey line) of fluorescence intensities of tdTomato-, MHC-II-, iNOS- and p22phox-immunostaining (below) of bone marrow-derived macrophages isolated from iNOS-tdTomato-cre mice and cultured for 48hrs with mCSF only (Ø), or with mCSF supplemented with LPS + IFNγ or with IL4 + IL13, n = 5 mice. Total number of cells analyzed for tdTomato: MØ = 1,381, M(LPS + IFNγ) = 2,082, M(IL4 + IL13) = 1,415; for MHC-II: MØ = 822, M(LPS + IFNγ) = 451, M(IL4 + IL13) = 607; for iNOS: MØ = 639, M(LPS + IFNγ) = 1,154, M(IL4 + IL13) = 583; for p22phox: MØ = 742, M(LPS + IFNγ) = 928, M(IL4 + IL13) = 832. Scale bar 20 µm. ***, P < 0.001, Kruskal-Wallis with Dunn’s post-hoc comparisons (p values for the Kruskal-Wallis tests and for the post hoc comparisons are p < 0.0001 for MHCII, p < 0.0001 for Tomato, p < 0.0001 for iNOS, p < 0.0001 for p22phox). (b) Confocal images of YFP-specific immunostaining (above, green) and quantifications (mean indicated as a grey line) of fluorescence intensity of YFP-, YM1-, iNOS- and CD206-specific staining (below) of bone marrow-derived macrophages isolated from Arginase-YFP mice and cultured for 48hrs with mCSF only (Ø), or with mCSF supplemented with LPS + IFNγ or with IL4 + IL13, n = 5 mice. Total number of cells analyzed for YFP: MØ = 948, M(LPS + IFNγ) = 702, M(IL4 + IL13) = 1,141; for YM1: MØ = 203, M(LPS + IFNγ) = 176, M(IL4 + IL13) = 342; for iNOS: MØ = 237, M(LPS + IFNγ) = 236, M(IL4 + IL13) = 241; for CD206: MØ = 280, M(LPS + IFNγ) = 129, M(IL4 + IL13) = 309. Scale bar 20 µm. ***, P < 0.001, Kruskal-Wallis with Dunn’s post-hoc comparisons (p values for the Kruskal-Wallis tests and for the post hoc comparisons are p < 0.0001 for iNOS, p < 0.0001 for EYFP, p < 0.0001 for CD206, p < 0.0001 for YM1 (MØ vs. LPS + IFNγ p = 0.0003, MØ vs. IL4 + IL13 p < 0.0001, LPS + IFNγ vs. IL4 + IL13 p < 0.0001).
