Anti cd86 bb515

Flow cytometric analyses were performed as previously described [36] . Briefly, MDMs were differentiated for five to six days and polarized as described above. 2.5-5 × 10⁵ cells were resuspended in FACSwash (PBS, 2.5% FCS, 0.1% sodium azide), blocked in human Fc Block solution (#564220, BD Biosciences, San Jose, CA, USA) for 15 minutes, and stained for 30 minutes on ice with anti-CD14-APC (#555399, BD Biosciences), anti-CD163-PE-CF594 (#562670, BD Biosciences), anti-CD80-BB515 (#565008, BD Biosciences) or anti-CD86-BB515 (#564544, BD Biosciences), and anti-HLA-DR-PerCP-Cy5.5 (#560652, BD Biosciences) antibodies. After washing, stained cells were resuspended in 1% paraformaldehyde in PBS prior to flow cytometric analysis on a BD LSRFortessa. Data were analyzed using BD FACSDiva software (BD Biosciences). Median fluorescence intensities of singlet cells were used to quantify surface marker expression.