Anti cd80 percp cy5

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.

The flow cytometry method was applied to determine the percentage and mean fluorescence intensity of CD11c, MHC II, CD80, and CD86 on the surface of living DCs. Cells were labeled with monoclonal antibodies conjugated with fluorochromes: anti-CD11c BV650, anti-MHC II APC-Cy7, anti-CD80 PerCP-Cy5.5, and anti-CD86 PE-Cy7 all from BioLegend. The cells were stained for 45 min at 4°C. To identify dead cells, 50 μl of DAPI dye solution (1 μg/ml, Molecular Probes) was added to the samples immediately before analysis. The expression of cell surface markers was analyzed using a BD LSRFortessa Cell Analyzer (Becton Dickinson, Cat. No. 649225B5) with the BD FACSDiva software version 8.0 and the NovoExpress software 1.3.0 (ACEA Biosciences, Inc.).

The following fluorescent-labeled monoclonal antibodies and staining reagents are used according to the manufacturer's specifications: anti-APC-CY7-live (BioLegend), anti-BV510-CD45 (BioLegend), anti-F4/80-BV421 (BioLegend), anti-CD11B-PE CY7 (BioLegend), anti-CD206-APC (BioLegend), anti-CD80-percp/Cy5.5 (BioLegend), anti-LY6C-APC (BioLegend), anti-CD11B-PE (BioLegend), anti-LY6G-PE CY7 (BioLegend), anti-CD3-PE CY7 (BioLegend), anti-CD4-BV421 (BioLegend), anti-CD8-percp/CY5.5 (BioLegend). The cells were proceeded by FACS Calibur flow cytometry and analyzed by the FlowJo software.