Gzmb clone qa16a02

For surface marker staining, cells were blocked with serum obtained from C57BL/6 mice at 4°C for 15 minutes, and then stained with fluorochrome-conjugated antibodies at 4°C for 20 minutes. The antibodies against CD45 (clone HI30), CD3 (clone OKT3), CD56 (clone 5.1H11), CD8α (HIT8α, clone RPA-T8), TCRVα7.2 (clone 3C10), CD45RA (clone HI100), HLA-DR (clone L243), CD4 (clone OKT4), CD69 (clone FN5D), γδTCR (clone B1), CCR7 (clone G043H7), and CD103 (clone Ber-ACT8) were purchased from BioLegend; the antibodies against CD45 (clone 2D1), CD122 (clone Mik-β3), and CXCR6 (clone 13B1E5) were purchased from BD Biosciences. For intracellular granzyme staining, cells were fixed and permeabilized with a Cytofix/Cytoperm Fixation/Permeabilization Kit (BD Biosciences) after surface marker staining, and then stained for GZMA (clone CB9, BioLegend), GZMB (clone QA16A02, BioLegend), and GZMK (clone GM26E7, BioLegend). Flow cytometric analysis was performed on a Cytek Aurora, and data were analyzed with FlowJo software (BD Biosciences).

After in vivo experiments, mouse tumors were rapidly excised and mechanically dissociated in PBS using scissors. The tumors were then digested in serum-free RPMI supplemented with 20 mg/ml DNase I (Roche), 20 mg/ml Dispase II (Roche) and 20 mg/ml collagenase I (Sigma) for 30–60 min at 37 °C with rotation to promote dissociation. The resulting single-cell suspensions were passed through 70 μM strainers twice, and red blood cells in the tumor samples were lysed with red blood cell lysis buffer (eBioscience, #00-4333-57) for 5 min at room temperature. Then, the single-cell suspensions were washed in Cell Staining Buffer (BioLegend) and incubated with the indicated flow antibodies at 4 °C for 30 min. Prior to staining with antibody panels, cells were blocked with a monoclonal antibody against CD16/32 (BioLegend) for 15 min at 4 °C. All the antibodies and reagents used for flow cytometry included ZombieRED (ECD, Biolegend, #423110), CD45 (clone 30-F11, Biolegend, #103116), CD3e (clone 145-2C11, Biolegend, #100328), CD4 (clone RM4-5, Biolegend, #100536), CD8 (clone 53–6.7, Biolegend, #100706), GZMB (clone QA16A02, Biolegend, #372208), PRF1 (clone S16009B, Biolegend, #154404), F4/80 (clone BM8, Biolegend, #123127), CD11b (clone M1/70, Biolegend, #101206), CD86 (clone GL-1, Biolegend, #105006) and CD206 (clone C068C2, Biolegend, #141719).