Cd80 v450

Macrophages cultured for 7 days were carefully harvested using Dispase enzyme solution and gentle scraping. Cells were counted and a minimum of 3 × 10⁵ cells were used for each FACS staining. Fc receptors were blocked in 3% BSA in 1× HBSS (Gibco) for 10 min. As this study focused on membrane proteins, permeabilization and fixation of cells were omitted. Instead, cells were re-suspended in PBS and incubated with the respective antibodies for 30 min. Antibodies used for FACS were αCD163-APC and αCD90-PE (both Biolegend), CD11b-V450, CD11c-PE, CD40-FITC, CD45-PE, CD209-Cy5.5, CD80-V450, CD86-V450, CD146-PE, and CD206-FITC (all BD Pharmingen). Unstained cells were used as control, as well as an IgG control. Cells were washed twice after antibody incubation and re-suspended in PBS for counting. Cell sorting was performed on a LSR-II instrument (BD Bioscience) using FACSDiva8 acquisition and analysis software. Cells were gated in three steps; first, cells were discriminated by size employing forward and side scatter (FSC and SSC, respectively). Second, doublets were removed by pulse-geometry gating the area and height of FSC. Lastly, the fluorescence signal of cells positive for respective markers was gated directly, plotting it against the SSC area.