Pe conjugated anti mouse ifn γ

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).