Pe conjugated anti mouse t bet

The lungs were removed 2 months after infection and digested with 75 U/ml collagenase (type 1; Sigma) at 37°C for 90 minutes. Isolated cells were filtered through a 20-μm nylon mesh and then stained with anti-CD4, anti-CD8, anti-CD3 and anti-TCRβ antibodies (Biolegend) to detect T cell subsets and analyzed by flow cytometry. T cell cytokine production was determined by flow cytometric intracellular cytokine analysis as described previously [18 (link)]. Briefly, cells were suspended at 10⁶/ml in RPMI 1640 containing 10% fetal calf serum, incubated with phosphomolybdic acid (50 ng/ml; Sigma) and ionomycin (500 ng/ml; Sigma) for 2 hours, and then incubated with brefeldin A (10 μg/ml; Sigma) for 2 hours at 37°C. Next, the cells were washed in PBS and fixed with 2% formaldehyde in PBS for 15 minutes at room temperature. The fixed cells were washed in PBS supplemented with 0.5% bovine serum albumin (BSA) and 0.02% sodium azide (PBS/BSA/azide). For intracellular cytokine detection, the cells were permeabilized with 0.5% saponin (Sigma) in PBS/BSA/azide, stained with PE-conjugated anti-mouse IFN-γ (Biolegend), PE-conjugated anti-mouse T-bet (eBioscience), PE-conjugated or APC-conjugated anti-mouse IL-17A (BD PharMingen), or APC-conjugated anti-mouse RORγT (eBioscience).

For cell surface and intracellular marker analysis, 10⁶ cells were incubated with fluorescent-conjugated antibodies in labeling solution (eBioscience, USA). The fluorescent-conjugated antibodies used in this study included PE-conjugated anti-mouse CD11c, FITC-conjugated anti-mouse CD11c, FITC-conjugated anti-mouse CD86, FITC-conjugated anti-mouse MHC-II, APC-conjugated anti-mouse CD80, PE-CY5-conjugated anti-mouse CD40, APC-conjugated anti-mouse CD4, FITC-conjugated anti-mouse IL-17A, FITC-conjugated anti-mouse Foxp3, PE-conjugated anti-mouse GATA-3, PE-conjugated anti-mouse T-bet antibodies, and FITC-conjugated anti-mouse CD103 (integrin alpha E) (all purchased from eBioscience, USA). Fluorescent-conjugated, isotype-matched, irrelevant antibodies were used to establish background fluorescein levels. Flow cytometry analysis was conducted on a FACSCalibur (BD Biosciences, USA), and FACSCalibur software (BD Biosciences) was used to analyze the flow data. DCs were gated for PE-CD11c, and then FITC-MHC-II expression and endocytic FITC-OVA levels were analyzed. MLR-lymphocytes were gated for APC-CD4, and then FITC-Foxp3 was analyzed.