Percp conjugated cd206

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).

The four materials (cp-Ti, Ti-SLA, Ti-NW and Ti-NW-Zn) samples were prepared and applied into 24-well plates and then RAW264.7 cells were seeded onto different topographies (1.5 × 10⁵ cells/well). Macrophage surface markers CD11c (M1) and CD206 (M2) were detected by flow cytometry for evaluating the different phenotypes. After 3-day incubation, cells were scraped off, centrifuged, and resuspended in purified anti-mouse CD16/32 (Biolegend, San Diego, CA, USA) for 10 min at 4 °C to block the nonspecific antigens. Then, RAW264.7 cells were incubated with PE-conjugated CD11c (Biolegend) and PerCP-conjugated CD206 (Biolegend) for 30 min at 4 °C. After washing twice in PBS, RAW264.7 cells were resuspended in PBS and analyzed on a flow cytometer (NovoCyte; ACEA Biosciences, San Diego, CA, USA). All samples were analyzed in triplicate.