Ly 6g gr1 percp 5.5 clone rb6 8c5

SM1 tumors and spleens were harvested from mice. Tumors were further digested with collagenase (Sigma-Aldrich). Splenocytes and cells obtained from digested SM1 tumors, were stained with Ab to CD3 BV605 (clone 17A2), Ly6C FITC (Clone AL-21), PD-L1/CD274 PE (Clone MIH5) (Becton Dickinson Biosciences, San Jose, CA), CD8a BV421 (Clone 53-6.7) (Biolegend, San Diego, CA), Ly-6G (Gr1) PerCP 5.5 (clone RB6-8C5), CD11b APC (clone M1/70), F4/80 Pacific blue/eFluor450 (clone BM8), CD25 APC (PC61.5), CD4⁺ FITC (RM4-5) (eBioscience, San Diego, CA), and analyzed with LSR-II or FACSCalibur flow cytometers (Becton Dickinson Biosciences), followed by analysis using Flow-Jo software (FLOWJO, LLC, Ashland, OR). Intracellular staining of Foxp3 PE (FJK-16s) (eBioscience) was done according to manufacturer's recommendations. After applying a gating strategy for the selection of the target population and exclusion of dead cells in tumors and spleens (Fig. S1A), the different immune cell populations were analyzed. Cells were analyzed with a LSR-II or FACSCalibur flow cytometers (BD Biosciences), followed by Flow-Jo software (Tree-Star, Ashland, OR) analysis as previously described.^{49 (link)}