Pe conjugated cd206 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.

For cell-surface analysis, cells were harvested by dissociation using 0.05% trypsin/EDTA. A total of 1 × 10⁶ cells were resuspended in 200 μL PBS with 1% FBS, incubated with antibodies at the recommended concentrations at 4 °C for 30 min, and then detected by flow cytometry (BD FACSCanto II, BD Biosciences, USA). Data were analyzed using FlowJo software. The antibodies used in flow cytometry assay were as follows: APC-conjugated CD163 antibody (333610, Biolegend), PE-conjugated CD206 antibody (321106, Biolegend), APC-conjugated CD44 antibody (559942, BD Biosciences), PE-conjugated CD24 antibody (555428, BD Biosciences).

The population analysis and sorting of Tregs, naive CD4⁺ T cells, CD8⁺ T cells, and TAMs were performed by flow cytometry using the FACSAria III flow cytometer (BD Biosciences) or NovoCyte flow cytometer (ACEA Biosciences). The antibodies used in flow cytometry assay were as follows: PE-Cy7-conjugated CD45 antibody (25-0451-82, eBioscience), FITC-conjugated F4/80 antibody (SC-71085, Santa Cruz Biotechnology), PE-conjugated CD206 antibody (141706, Biolegend), PE-conjugated CD4 antibody (100407, Biolegend), APC/Cyanine7-conjugated CD8a antibody (100714, Biolegend), PE-Cy7-conjugated CD25 antibody (25-0251-81, Invitrogen), APC-conjugated FOXP3 antibody (17–4777-42, eBioscience), and BV650-conjugated CD45RA antibody (564360, BD Biosciences). The sorted lymphocytes were re-suspended in RPMI-1640 complete culture medium and stimulated by 50 ng/ml PMA, 1 ug/ml ionomycin, and 10 ng/ml IL-2 (Multiscience, Hangzhou, China) in vitro for 24–48 h, and then continuously cultured in RPMI-1640 complete culture medium.