Anti ly6g bv605

All antibody staining was preceded by 15 min of 1:50 FcγR block in FC buffer, on ice. Extracellular antibodies were then added to FC buffer containing FcγR block, and incubated for 45 min on ice. Intracellular staining was accomplished after surface staining using the Foxp3 staining kit (eBioscience). Myeloid subsets were stained with anti-CD1lb-FITC, anti-Ly6C-AF700, anti-MR-PE-Cy7, anti-CD11c-PE/Dazzle594, anti-Ly6G-BV605 (BioLegend), anti-MHCII-eFluor450 (eBioscience), and anti-F4/80-APC (BioRad). To evaluate Tregs, cells were stained with anti-CD3-AF488, anti-CD4-APC, anti-CD25-PerCP-Cy5.5 (BioLegend), and anti-Foxp3-PE (BD Pharmingen). To assess T cell activation, total tumor cells were incubated for 4 hr at 37°C in a 5% CO2 incubator with Protein Transport Inhibitor Cocktail containing brefeldin A and monensin, or with Cell Stimulation Cocktail containing protein transport inhibitors and PMA/ionomycin (eBioscience). Cells were then stained with anti-CD3-AF488, anti-CD4-PE, and anti-CD8-PerCP-Cy5.5 followed by intracellular stain with anti-IFNγ-APC (BioLegend). In addition, 1E5 CD3⁺ cells were stimulated with 4E4 CD3/CD28 Dynabeads (ThermoFisher) for 3 days, followed by staining using anti-CD3-PerCP-Cy5.5, anti-CD8-BV650, anti-CD4-BV605, anti-IFNγ-APC, anti-TNFα-BV421 (BioLegend), and anti-GranzymeB-PE (eBioscience).