Anti ifnγ pe cf594

M-MDSC and G-MDSC were phenotyped using flow cytometry. Tumor was cut into small fragments followed by digestion with tumor disassociation kit for 30 min (Miltenyi Biotec, USA), and then filtered by 70 µm cell strainers. Mononuclear cells was enriched by subjecting the single cell suspension to percoll gradient. Briefly, 70% percoll (4 ml) was added to the centrifuge tube, 30% percoll (4 ml) was carefully layered, and then tumor suspension (4 ml) was carefully layered on the top. After centrifuging at 1500 rpm for 15 mins, the buffy coat layer in the middle was collected and washed with PBS, followed by staining with anti-CD4 FITC, anti-CD8 Percp, anti-Ly6G AF700, anti-Ly6C APC, anti-CD11b APC-Cy7, anti-IFNγ PE-CF594, and anti-granzyme B PE antibodies, along with appropriate isotype controls (all from BD) for flow cytometry analysis (BD FACSCalibur). M-MDSC were defined as CD11b⁺Ly6C⁺Ly6G^- and G-MDSC were defined as CD11b⁺Ly6G⁺Ly6C^-.

Tumor tissue was harvested and cut into small fragments followed by digestion with tumor disassociation kit for 30 min (Miltenyi Biotec, USA), and then filtered by 70 µm cell strainers. Mononuclear cells were enriched by percoll gradient centrifuging of the single cell suspension. Cells were washed with PBS and stained with Live/Dead dye, anti-CD4 FITC, anti-CD8 PE-Cy7, anti-Ki67 APC, anti-IFNγ PE-CF594, anti-FoxP3 Percp and anti-granzyme B PE antibodies, along with appropriate isotype controls (all from BD) for flow cytometry analysis (BD FACSCalibur).