Pacific blue conjugated anti

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).