Fitc conjugated anti cd8 mab

To analyze tumor‐infiltrating immune cells, tumor tissues were resected from mice individually, 22 days after tumor inoculation (4 days after the second chemotherapy treatment). The tumors were minced, placed on glass slides, and passed through gauze mesh and nylon mesh immediately before flow cytometry. The following mAbs were used: APC‐conjugated anti‐CD45 mAb (BioLegend), FITC‐conjugated anti‐Gr‐1 mAb (BioLegend), PE‐conjugated anti‐CD11b mAb (BioLegend), FITC‐conjugated anti‐CD8 mAb (BioLegend), PE‐conjugated anti‐CD4 mAb (BioLegend), and PE/cy7‐conjugated anti‐Ly6C mAb (BioLegend). Similarly, spleen cells of tumor‐bearing and treated mice were analyzed. Analysis was performed using FACSCalibur (Becton‐Dickinson).

A lung tissue single cell suspension was prepared as previously [11 (link)]. The cells from lung tissues were then stimulated by phorbol myristate acetate (50 ng/ml) (Sigma, MO, USA) and ionomycin (1000 ng/ml) (Sigma, MO, USA) for 5 h. Brefeldin A (50 ng/ml) (Sigma, MO, USA) was used to block the flow of cytokines from cytoplasm. Then, the cells were incubated with the appropriate concentration of PE-CY5 conjugated anti-CD3 mAb (Biolegend, San Diego, CA) and FITC-conjugated anti-CD8 mAb (Biolegend, San Diego, CA) for 30 min at 4°C in darkness. Then the cells were fixed and permeabilized with Cytofix/Cytoperm solution (BD Biosciences, San Jose, CA) for intracellular cytokine staining. After incubation with PE conjugated anti-IL-17A mAb (Biolegend, San Diego, CA) for 30 min at 4°C in darkness, flow cytometry was performed on a BD LSR II cytometer to determine the percentages of Tc17 (CD3⁺CD8⁺IL-17⁺) or Th17 (CD3⁺CD8^-IL-17⁺) lymphocytes.