Cd68 fitc

All data were acquired using a Cytomics FC 500 flow cytometer operating on CXP software equipped with a dual 488 nm/635 nm (blue/red) laser (Beckman Coulter, Brea, CA, USA). Intracellular staining and staining of cell surface antigens were performed simultaneously using the MACS Inside Stain Kit (Miltenyi Biotec, Bergisch Gladbach, Germany). Briefly, Inside Fix buffer was added to 1 × 10⁵ cells and then resuspended in PBS. The solution was incubated at room temperature and pressure for twenty minutes, after which the cells were washed twice with PBS via centrifugation for five minutes at 300 g. The supernatant was discarded, and the pellet was resuspended in 100 µL of Inside Perm buffer; cells were stained with fluorochrome-conjugated antibodies at appropriate dilutions. Cells were incubated for fifteen minutes at room temperature and then washed with Inside Perm buffer. The supernatant was discarded, the pellet was resuspended in 400 µL of PBS, then flow cytometric analysis was performed. Antibodies against CD11b-VioBright FITC, CD86-PE-Vio770, and CD68-FITC were obtained from Miltenyi Biotec, (Bergisch Gladbach, Germany), whilst antibodies against CD206-PE and CD163-PE were obtained from eBioscience (San Diego, CA, USA).