Apc anti mouse cd206 mmr

To analyze M1/M2 polarization of macrophage cells, macrophages equilibrated with DMEM were treated with lipopolysaccharide (0127:B8; 50 ng/ml; Sigma-Aldrich) for 4 h or IL-4 (0.1 ng/ml; R&D system) for 24 h. These macrophages were incubated with FITC anti-mouse F4/80 (F480, eBioscience), PE anti-mouse CD80 (B7-1; eBioscience), APC anti-mouse CD86 (B7-2, eBioscience), and APC anti-mouse CD206 (MMR, Biolegend) (Additional file 2) for 30 min at 4 °C. After washing twice with 2 % FBS in PBS, macrophages were used for flow cytometry analysis. Data were collected on a FACS Calibur flow cytometer (BD Biosciences) and analyzed using Cell Quest Pro software (BD Biosciences). Alteration in protein expression of M1 (iNOS) and M2 marker (arginine-1) was analyzed using protocol as described in 2.11. Western blot analysis.

Flow cytometry was conducted as previously reported [40 (link)]. Briefly, the colonic tissues were minced and incubated in Hank’s balanced salt solution (HBSS) containing 5 mM EDTA and 1 mM DTT at 37°C for 30 min to dissociate the epithelial cells. After filtration through a 100-μm cell strainer, the remaining pieces were incubated with an RPMI medium containing 0.05% collagenase D (Roche, Shanghai, China) and 0.05% DNase I (Roche, Shanghai, China) for 30 min with gentle shaking. As for J774A.1 cell samples, about 1 × 10⁶ cells per sample were collected after experimental treatment. Cell suspensions were then passed through a 70-μm cell strainer and collected for subsequent flow cytometry analysis. Lamina propria mononuclear cells (LPMCs) or J774A.1 cells was stained with the following antibodies: APC anti-mouse/human CD11b (Biolegend/M1/70), PE anti-mouse CD11c (Biolegend/N418), PE anti-mouse F4/80 (Biolegend/BM8), and APC anti-mouse CD206 (MMR) (Biolegend/C068C2). CD11b⁺CD11c⁺ cells were considered M1 macrophages, while F4/80⁺CD206⁺ cells were treated as M2 macrophages [40 (link)].