Pe conjugated cd163 antibody

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.

Macrophages were incubated with FITC-conjugated F4/80 antibody (SC-71085, Santa Cruz, Dallas, TX, USA) and PE-conjugated CD163 antibody (12-1639-42, eBioscience, San Diego, CA, USA) for 30 min at 37 °C. The F4-80⁺/CD163⁺ subpopulation was quantified by flow cytometry using the FC500 flow cytometer (Beckman Coulter, California, USA) and defined as M2 phenotype macrophages (TAMs).