Percp cy5.5 conjugated anti il 17a

Cells were harvested and stimulated for 4 h with PMA (50 ng/ml), ionomycin (750 ng/ml), and Brefeldin A (BioLegend). For intracellular staining, cells were fixed and permeabilized (eBioscience) and then stained with FITC-conjugated anti–IFN-γ (505806, BioLegend), PE-conjugated anti–IL-13 (12-7133-81, eBioscience), PerCP/Cy5.5-conjugated anti–IL-17A (506919, BioLegend), and APC-conjugated anti-FOXP3 (17-5773-80, eBioscience) antibodies. To analyze lymphocyte development in Pten^fl/flIl17a^cre mice, cells isolated from the thymus, pLNs, and spleen were stained with FITC-conjugated anti-CD3ε (11-0031-81, eBioscience), PE-conjugated anti-B220 (12-0451-82, eBioscience), PerCP/Cy5.5-conjugated anti-CD8 (126609, BioLegend), APC-conjugated anti-CD4 (100411, BioLegend), FITC-conjugated anti-CD44 (103006, BioLegend), PE-conjugated anti-CD62L (104407, BioLegend), and PerCP/Cy5.5-conjugated anti-TCR γ/δ (118117, BioLegend) antibodies. For other surface antigen staining, cells were stained with APC-conjugated anti–IL-6Rα (115811, BioLegend), APC-conjugated anti-CD45 (103111, BioLegend), FITC-conjugated anti–GR-1 (108405, BioLegend), and FITC-conjugated anti-CD11b (101205, BioLegend) antibodies. Stained cells were then analyzed using a FACSCalibur flow cytometer (BD Bioscience), and data were analyzed with FlowJo software.

Mice were anesthetized and perfused. The brain and spinal cord were then removed and homogenized. Mononuclear cells were isolated by gradient centrifugation at 670 g for 30 min on a 30/70% Percoll gradient (GE Healthcare). Isolated cells were stimulated with PMA (50 ng/ml), ionomycin (750 ng/ml), and Brefeldin A (BioLegend) for 4 h and then stained with FITC-conjugated anti–IFN-γ (BioLegend), PerCP/Cy5.5-conjugated anti–IL-17A (BioLegend), FITC-conjugated anti–GM-CSF (505403, BioLegend), APC-conjugated anti-FOXP3 (eBioscience), and APC-conjugated anti-CD4 antibodies (Lee et al., 2015 (link)).