Macrophage Phenotype Evaluation Protocol

The surface markers CCR7 (M1) and CD206 (M2) were examined via flow cytometry for evaluating different macrophage phenotypes. The specific procedure is the same as described in previous studies [5 (link)]. Anti-mouse CD16/32 (Biolegend, USA) was used to block the non-specific antigens. The antibodies for flow cytometry in this study included PE-conjugated CCR7 and PerCP-conjugated CD206 (Biolegend, USA), and the isotype controls were PE-conjugated Rat IgG2a, ĸ and PerCP-conjugated Rat IgG2a, ĸ (Biolegend, USA). The expression level of M1/M2 macrophage markers of all samples was analyzed using a flow cytometer (NovoCyte, ACEA Biosciences, USA) in triplicate. In addition, the surface markers IL-1β (M1) and CD163 (M2) were detected via western blot for further evaluating different macrophage phenotypes. The first antibodies for western blot were anti-IL-1β (ABclonal, Wuhan, China, the dilution was 1:1000), anti-CD163 (ABclonal, the dilution was 1:1000) and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (ABclonal, the dilution was 1:4000).