Tumor Immune Profiling via Flow Cytometry

At the end of the animal experiments, tumors were harvested, mechanically cut into small pieces, and digested into a single-cell suspension using a mouse tumor dissociation kit (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer’s instructions. Dead cells isolated from tumors and draining lymph nodes (DLNs) were stained using the Fixable Aqua Dead Cell Stain Kit (Invitrogen). Cell surface/intracellular markers were stained, and immune phenotypes in the tumors and DLNs were analyzed as previously described [19 (link)]. The following fluorophore-conjugated antibodies were used in this analysis: efluor780 anti-mouse CD45, peridinin chlorophyll protein-cyanine 5.5 anti-mouse CD4, efluor450 anti-mouse CD8, allophycocyanin (APC) anti-mouse Tim3 (eBioscience), fluorescein isothiocyanate (FITC) anti-mouse CD3, phycoerythrin (PE) anti-mouse FoxP3, and APC anti-mouse IFNγ (BioLegend).