Phenotypic characterization of tumor infiltrating and lymph node cells by flow cytometry

Tumors were harvested from tumor bearing mice and minced using razor blades followed by digestion with accutase (PAA), collagenase IV (Worthington), hyaluronidase (Sigma), and DNAse type IV (Sigma) for 60 min at 37 °C with constant shaking. The cell suspensions were filtered using a cell strainer (70 μm). Lymph node cells were isolated by mashing using the end of a 1 mL syringe. Cells were filtered through a 70 μm nylon mesh. Red blood cells (RBCs) were lysed using RBC lysis buffer (eBioscience). Single cell suspensions derived from tumor and lymph nodes were blocked with rat anti-mouse FcγIII/II receptor (CD16/CD32) blocking antibodies (“Fc-Block”) and stained with live/dead cell-exclusion dye (Zombie UV dye; Biolegend). The cells were then incubated with fluorophore-conjugated antibodies directed against cell surface antigens, washed and resuspended in FACS buffer (PBS + 2%FBS). For intracellular antigens (FoxP3), cells stained with cell surface antibodies were fixed (IC fix, eBioesceince) and permeabilized (Perm buffer; eBioscience) prior to incubation with antibodies directed against intracellular antigens. Cell populations were analyzed on a BD Fortessa. Cells were discriminated using the following combination of cell markers after gating on single cells (discriminated by FSC-A and FSC-H) and excluding non-viable cells (Live/Dead negative). TAMs were denoted by CD45⁺CD11b⁺Ly6C⁻Ly6G⁻F4/80^high. G-MDSC were CD45⁺CD11b⁺Ly6G⁺ and M-MDSC were CD45⁺CD11b⁺Ly6C⁺. CD4⁺/CD8⁺ T cells were CD45⁺CD11b⁻CD3⁺CD4⁺ or CD8⁺. Tregs were FOXP3⁺CD25⁺CD4⁺ T cells. B cells were CD45⁺CD11b⁻CD3⁻CD19⁺. Tumor cells were noted as CD45⁻.