2.8. Tumor-Infiltrating Immune Cell Analysis Using Flow Cytometry

For flow cytometry analysis, tumors were harvested and dissociated into a single-cell suspension by 1-h incubation at 37 °C in digestion buffer containing 2 mg/mL collagenase D (COLLD-RO, Merck, Kenilworth, Darmstadt, Germany) and 40 μg/mL DNase I (10104159001, Merck). The cell clumps were removed through a 70-μm cell strainer (352350, Corning, NY, USA) and a 40-μm nylon mesh. The red blood cells were removed by incubation for 3 min at RT in ACK lysis buffer (A1049201, Fisher Scientific, Waltham, MA, USA). To exclude dead cells, the dissociated cells were stained with Fixable Viability Dye eFluorTM 450 (65-0863-18, Invitrogen) on ice for 30 min before antibody staining. After washing with FACS buffer (1% fetal bovine serum in PBS), the cells were incubated on ice for 30 min in FACS buffer with the following antibodies: FITC-conjugated anti-CD11b (rat, clone M1/70, Invitrogen), FITC-conjugated anti-CD4 (rat, clone RM4-5, Invitrogen), Alexa Fluor 488-conjugated anti-mouse iNOS (rat, clone CXNFT, Invitrogen), PE-conjugated anti-mouse CD8a (rat, clone 53-6.7, Invitrogen), PE-conjugated anti-mouse CD86 (rat, clone B7-2, Invitrogen), PE-conjugated anti-mouse F4/80 (rat, clone BM8, Invitrogen), PerCP/Cy5.5-conjugated anti-mouse Ly-6C (rat, clone HK1.4, Invitrogen), PerCP/Cy5.5-conjugated anti-mouse CD45 (rat, clone 30-F11, Invitrogen), APC-conjugated anti-mouse CD3 (rat, clone 17A2, Invitrogen), APC-conjugated anti-mouse CD11c (armenian hamster, clone N418, Invitrogen), APC-conjugated anti-mouse Ly-6G (rat, clone 1A8-Ly6g, Invitrogen), Alexa Fluor 700-conjugated anti-mouse Arginase 1 (rat, clone A1exF5, Invitrogen), APC/eFluor 780-conjugated anti-mouse CD11b (rat, clone M1/70, Invitrogen), APC/Fire750-conjugated anti-mouse CD80 (armenian hamster, clone 16-10A1, BioLegend), or eFluor 506-conjugated anti-mouse CD45 (rat, clone 30-F11, Invitrogen). We analyzed the following cell subsets: M1-like TAMs, gated as viability dye⁻/CD45⁺/CD11b⁺/Ly-6G⁻/Ly-6C⁻/F4/80⁺/iNOS⁺/Arg1⁻ cells; M2-like TAMs, gated as viability dye⁻/CD45⁺/CD11b⁺/Ly-6G⁻/Ly-6C⁻/F4/80⁺/iNOS⁻/Arg1⁺; TILs, gated as viability dye⁻/CD45⁺/CD3⁺/CD8⁺ or CD4⁺ cells; activated DCs, gated as viability dye⁻/CD45⁺/CD11c⁺/CD86⁺ or CD80⁺ cells. The stained cells were analyzed using a CytoFLEX flow cytometer (Beckman Coulter, Brea, CA, USA), and the data were analyzed with FlowJo software (Tree Star, San Carlos, CA, USA).