Flow cytometry canto 2 flow cytometer

Single-cell suspensions were obtained from mouse tumors after tumor disaggregation as described in the previous method. The cells were treated with anti-CD16/CD32 antibodies to block Fc receptors. Subsequently, the cells were stained with the following antibodies (BioLegend): Brilliant Violet 510-conjugated anti-CD45, FITC-conjugated anti-CD3, APC-Cy7-conjugated anti-CD8a, FITC-conjugated anti-CD8a, PerCP-conjugated anti-PD-1, APC- conjugated anti-CD44, Pacific Blue-conjugated anti-CD69, PE-conjugated anti-KLRG-1, PerCP-conjugated anti-CD25, APC- conjugated anti-CD103, APC-Cy7-conjugated anti-CD4, Pacific Blue-conjugated anti-FOXP3, PerCP-conjugated anti-MHC-II, APC-Cy7-conjugated anti-CD11c, Pacific Blue-conjugated anti-CD11b, PE-conjugated anti-XCR-1. The cells were stained for 30 min at 4 °C in the dark. For intracellular staining, cells were fixed using 4% Paraformaldehyde Phosphate Buffer Solution (Wako, Osaka, Japan) and permeabilized using 0.5% Polyoxyethylene (10) Octylphenyl Ether (Wako, Osaka, Japan) then stained with antibody for 30 min at 4 °C in the dark. After extensive washing with FACS buffer, the cells were subjected to flow cytometry Canto II flow cytometer (BD Biosciences, San Jose, CA). Data were analyzed using FlowJo software (BD Biosciences, version 10.6).

Cultured cells were incubated for 20 min at 4 °C with appropriate antibodies APC-conjugated anti-BDCA-4, FITC-anti-CD123 (Miltenyi, Bergisch Gladbach, Germany), PE.cy7-anti-CXCR4 clone 12G5 (Biolegend, San Diego, CA), Live/Dead Green Kit (ThermoFisher Scientific) or with appropriate isotype-matched control antibodies (5μg/mL each) in PBS containing 2% mouse serum (Sigma, Saint Louis, MO) and FC-receptor blockers (BD Biosciences, San Jose, CA). For IRF-7 intracellular staining, cells were fixed with 2% PFA then permeabilized with 0.5% saponin before being stained for 30 minutes at 4 °C with anti-IRF-7 antibody (BD Biosciences). Flow cytometry analysis was performed on a flow cytometry Canto II flow cytometer using flow cytometry Diva software (BD Biosciences, San Jose, CA). FlowJo software (Treestar, Ashland, OR) was used to analyze data.