Fig. 5

a-GalCer-treated mice show a higher frequency of M1 macrophages and low tumor growth. Naïve C57BL6 mice were given s.c. injection of B16F10 cells (1 X 10⁶ cells/mouse) and animals were treated with α-GalCer injection (2 μg/mouse/i.p injection) on the day + 1, + 5, + 10, and + 15 (day with respect to tumor cell injection). a At day 20, the percentage of F4/80⁺CD11b⁺ cells in the spleen and tumor were analyzed. n = 3–5 mice/group. b At day 20, the percentage of iNOS⁺ cells (M1 macrophage; left panel) and CD206⁺ cells (M2 macrophage; right panel) in the spleen were analyzed after gating F4/80⁺CD11b⁺ cells. n = 3–5 mouse/group. c iNOS⁺F4/80⁺ cells (M1 macrophage) in the spleen and tumor were analyzed by immunofluorescence microscopy and the representative images are shown. Original magnification 400x. d CD206⁺F4/80⁺ cells (M2 macrophage) in the spleen and tumor of DMSO and α-GalCer-treated mice were analyzed by immunofluorescence staining (upper panel). Original magnification 400x. Representative contour plots of CD206⁺F4/80⁺ cells (M2 macrophage) are shown (lower panel). e F4/80⁺ cells were depleted by i.v. injection of anti-F4/80 mAb or anti-Gr1 mAb on the day − 1, + 5, + 10 and + 15 with respect to tumor cell injection. Along with F4/80⁺ cell depletion, α-GalCer (2 μg/mouse/injection) was given on the day + 1, + 5, + 10, + 15 and + 20. Tumor growth was monitored, and the tumor area was calculated and plotted. n = 4–5 mice/group. f At day 20, IFN-γ expression in the splenic NKT cells were analyzed and plotted. n = 3–5 mice/group. The bar represents s.e.m., and each dot represents an individual mouse. (a, b, f). One-way ANOVA (e). Student’s t-test (a, b, f). *p < 0.05; **p < 0.01; ns, not significant