Sc 71085

For the phenotype analysis of Raw264.7 macrophages, cells were first treated as indicated. Then, Raw264.7 cells were harvested and incubated with FITC-conjugated F4/80 antibody (SC-71085, Santa Cruz, CA, USA), PE-conjugated CD206 antibody (141,705, Biolegend, CA, USA), or PE-Cy7-conjugated CD206 antibody (E-AB-F1135H, Elabscience, Houston, TX, USA) for 30 min at 37 °C. For the phenotype analysis of THP1 macrophages, cells were treated as indicated, harvested and incubated with APC-conjugated F4/80 antibody (17-4801-82, Invitrogen) and PE-conjugated CD163 antibody (12-1639-42, eBioscience) for 30 min at 37 °C. For the phenotypic analyses of primary macrophages isolated from mouse 4 T1-Luc xenografts, cells were incubated with CD45-PE-Cy7 (25-0451-82, eBioscience), F4/80-APC antibody (17-4801-82, Invitrogen), and CD206-PE antibody (141,705, Biolegend) for 30 min at 37 °C. For PD-L1 expression analyses of primary macrophages, cells were incubated with CD45-PE-Cy7 (25-0451-82, eBioscience), F4/80-FITC antibody (SC-71085, Santa Cruz), and PD-L1-APC antibody (124,312, Biolegend) for 30 min at 37 °C. After incubation, the cells were washed once with PBS and subjected to flow cytometry analysis.

The phenotype of macrophages was identified by analyzing the surface markers of macrophages using flow cytometer. Briefly, macrophages were harvested, washed, and resuspended in 100 µl PBS solution at a density of 5 × 10⁶ cells/ml. For Thp1 macrophages, cells were incubated with FITC-conjugated F4/80 antibody (SC-71085, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and PE-conjugated CD163 antibody (12-1639-42, eBioscience, San Diego, CA, USA) for 30 mins at 37°C. For Raw264.7 macrophages, cells were incubated with FITC-conjugated F4/80 antibody (SC-71085, Santa Cruz) and PE-conjugated CD206 antibody (141705, Biolegend, San Diego, CA, USA) for 30 mins at 37°C. For phenotype analysis of primary macrophages extracted from mouse 4T1-Luc xenografts, cells were incubated with CD45-PE-Cy7 (25-0451-82, eBioscience), FITC-conjugated F4/80 antibody (SC-71085, Santa Cruz) and PE-conjugated CD206 antibody (141705, Biolegend) for 30 mins at 37°C. After incubation, cells were washed once with PBS and subjected to analysis using a FC500 flow cytometry (Beckman Coulter, Fullerton, CA, USA) or a FACSAria III flow cytometer (BD Biosciences, San Diego, CA, USA). The F4/80⁺/CD163⁺ subpopulation, the F4/80⁺/CD206⁺ subpopulation or the CD45⁺/F4/80⁺/CD206⁺ subpopulation were quantified and defined as M2 phenotype macrophages.