Anti mhc 2 bv421

From each group of animals, 1–2 million cells aliquots were prepared and to exclude dead cells from analysis, cells were first stained with the fixable viability dye eFluor 780 (eBioscience) for 15 min at room temperature (RT). Cells were incubated with anti-mouse CD16/CD32 Fc Block antibody (BD Biosciences, San Jose, CA), for 20 min at 4°C. Cells were then stained for 30 min at RT with appropriately diluted PE-conjugated 5-OP-RU-loaded species-specific MR1-tetramers or α-GalCer (PBS-44)–loaded CD1d tetramer conjugated to APC, anti-CD3-FITC (BioLegend), anti-CD161-BV510 (BioLegend), anti-CD49b-BV711 (BD), anti-TCRγδ-PE-Cy7 (BioLegend), anti-TCRβ-BV421 (BioLegend), anti-CD45R-PE-Cy5, and anti-CD44-BV650 (BioLegend). To evaluate different antigen-presenting cells, after Fc Block incubation, cells were also surface stained with anti-CD45 AF700 (BioLegend), anti-CD11b-FITC (BioLegend), anti-CD11c-PE Cy-7 (BD), anti-Siglec-F- BV711 (BD), anti-CD64-BV605 (BioLegend), anti-CD24-PE (BioLegend), anti-Ly-6G-PerCP-Cy5.5 (BioLegend), anti-CD103-BV510 (BioLegend), anti-CD86-APC (BioLegend), anti-Ly6C (PE dazzle 594), and anti-MHC II BV421 (BioLegend) for 30 min at 4°C. Total 10⁶ gated events per sample were collected using Fortessa flow cytometer (Becton Dickinson, San Diego, CA), and results were analyzed using FlowJo 10.4.2 software.

After collecting cultured cells and blocking with rat serum for 30 min, the following flow cytometry antibodies were used for phenotypic analysis: anti-CD11c-APC (Biolegend, California, USA), anti-MHCII-BV421 (Biolegend, California, USA), anti-CD80-Percp/cy5.5 (Biolegend, California, USA), anti-CD86-PE (Biolegend, California, USA), anti-CD40-PE/Cy7 (Elabscience, Wuhan, China). Data was acquired using FACS Canto II (BD Biosciences, USA) and analyzed using FlowJo software.