Tumor Immune Cell Analysis by Flow Cytometry

Prepared for single cell suspensions: for the murine samples, subcutaneous tumor mice or bone tumor mice were sacrificed by CO₂. Blood samples were drawn from tumor mice heart, and tissues of spleens, femurs, and tumors were extracted and minced by scissor. Femurs and tumors need to be digested with collagenase IV (Sigma-Aldrich, 0.2%) for 30 min at 37 °C and were terminated by RMPI 1640 (serum). Suspensions was strained through a 70-μm cell strainer to obtain a single-cell fluid. Red blood cells were lysed by lysis buffer (BD, 555899).
Antibody incubation and examination: 1 × 10 ⁶ cells from single cell suspensions were labeled with fluorescence-conjugated antibodies: APC-CD11b (Clone: M1/70; eBioscience™), PE-Ly6G (Clone: 1A8; MilliporeSigma™), Fitc-CD4 (Clone: GK1.5; eBioscience™), APC-CD8 (Clone: 53–6.7; R&D Systems™), PE-Perforin (Clone: eBioOMAK-D; eBioscience™), PerCP-5.5-Granzyme (Clone: NGZB; eBioscience™), PerCP-5.5-Arginase (Polyclonal, Novus Biologicals™), and PE-iNOS (Clone: CXNFT, eBioscience™), PE-EpCAM (Clone: EBA-1, BD Biosciences). Cells were staining for 30 min at 4 °C and washed with PBS twice before examined. For intracellular protein markers (arginase, iNOS, granzyme, and perforin), cells were conducted with fixation/permeabilization procedure following the instruction (BD Biosciences, 555028) before being stained with fluorescently labeled antibodies for 30 min. Cells labeled with fluorescence-conjugated antibodies were then examined with BD LSR Fortessa Analyzer and analyzed with FlowJo v10.6.2. Prostate cancer cells labeled with PE-EpCAM were isolated with FACS Aria sorting system for following experiments.