70 mm nylon cell filter

The mouse tumor was cut into 2- to 3-mm pieces to make a single cell suspension. The tumor and spleen were mechanically macerated and passed directly through a 70-mm nylon cell filter (Corning). The cells were washed with flowing buffer (0.1% BSA in PBS) and incubated with Zombie NIRTM (Biolegend, 1:100) for 20 min at room temperature, and then cells were stained with extracellular antibodies: anti-mouse CD3 FITC, anti-mouse CD8a APC, anti-mouse CD4 PerCP/Cy5.5, anti-mouse Ly-6G/Ly-6C (Gr-1) PE/Cy7, anti-mouse CD11b PE, anti-mouse CD 279 (PD-1) Brilliant Violet 421TM or anti-mouse CD45 Brilliant Violet 510TM (Biolegend). For intracellular staining, cells were stained using the FoxP3/T-bet/IFN-γ/granzymeB (GrzmB) staining buffer set (Biolegend) for fixation and permeabilization after completion of extracellular staining according to the manufacturer’s protocol. Cells were then stained with Biolegend’s anti-mouse FoxP3 PE, anti-mouse T-bet PE/Cy7, anti-mouse GrzmB FITC or anti-mouse IFNr bright purple 510TM. All data were collected on a flow cytometer (BD Biosciences, Canto II) and analyzed using FlowJo v10 software (Tree Star, Inc.)