Fig 1: An hFL-OPG-Fc treatment induced M2 macrophage polarization in vitroMurine macrophage cells were stimulated with LPS (1,000 ng/mL) followed by a 24-h treatment with either PBS, hFL-OPG-Fc (200 ng/mL), or both IL-4 and IL-13 (10 ng/mL of each) for M2 phenotype polarization. (A) To test whether hFL-OPG-Fc treatment induces M2 polarization, total macrophage and M2 phenotypes were labeled using F4/80 and CD206, respectively, and sorted by flow cytometry. (B–D) hFL-OPG-Fc treatment induced a slight increase in total macrophages (F4/80+ cells) (B), a marked decrease in M1 macrophages (F4/80+/CD206−) (C), and a significant increase in M2 macrophages (F4/80+/CD206+) (D) compared to PBS-treated cells. (E and F) IL-4/IL-13-treated cells were used as a positive control for M2 polarization. The experiment was performed four separate times. Western blot results show that hFL-OPG-Fc significantly decreased TNF-α (E) and iNOS (F) proteins levels, both M1 macrophage markers. (G) The expression of chitinase 3-like 3 and 4 proteins (Ym1/2), an M2 macrophage marker, was significantly increased in hFL-OPG-Fc-treated cells. Protein levels were normalized to GAPDH content and expressed in ratio from PBS (n = 7–15). Data are expressed as means ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001 indicate significantly different from PBS using one-way ANOVA for multiple comparisons followed by a Dunnett’s post hoc test.
Fig 2: Sequential release of IL-10 attenuates the inflammatory response in DWs. (A) IF staining of Macrophages in the skins at 3 days post-treatment, scale bar = 50 μm. (B) Quantification of F4/80+iNOS+ M1 and F4/80+CD206+ M2 cells. (C, D) WB and quantification analysis of skin tissue iNOS and CD206 protein levels at day 3 after treatment. (F) qRT-PCR analysis of skin tissue IL-1β, TNF-α, Arg-1, and IL-10 mRNA levels at day 3 after treatment. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 vs PBS. #P < 0.05, ##P < 0.01, ###P < 0.001, ####P < 0.0001 vs AgILgel.
Supplier Page from Abcam for Anti-iNOS antibody [EP16634]