Mouse th1 th17 phenotyping kit

Antibodies for detection of the following targets were purchased as indicated: caspase-3 from Cell Signaling Technology; Bax (ab7977) from Abcam; Bcl-2 from BD Biosciences-Pharmingen; and actin from Sigma. Phorbol 12-myristate 13-acetate (PMA), ionomycin, and OVA were purchased from Sigma. Mouse Th1/Th17 phenotyping kit and Perm/Fix solution were purchased from BD Biosciences (USA). IFN-γ and IL-17A ELISA kits were obtained from eBioscience (USA). FITC-anti-CD4 was obtained from Sungene Biotech (Tianjin, china). The MOG35-55 peptide (MEVGWYRSPFSRVVHLYRNGK) was synthesized by Chinese Peptide Company (Hangzhou, China). DyLight 800/DyLight 680-conjugated secondary antibodies against mouse or rabbit IgG were purchased from Rockland Immunochemicals (USA). Recombinant human PDCD5 protein was supplied by Beijing Biosea Biotechnology Co. The endotoxin activity of the rhPDCD5 protein received was <10 EU/mg as detected using the limulus amebocyte lysate assay, and the purity of the rhPDCD5 protein was >95 %.

Lymph node and CNS mononuclear cells were isolated and stimulated with phorbol 12-myristate 13-acetate (50 ng/ml) (Sigma-Aldrich) and ionomycin (1 µg/ml) (Sigma-Aldrich) for 5 h at 37°C in RPMI 1460 medium containing 10% FBS and GolgiSTOP (BD Biosciences, San Diego, CA, USA). IL-17 and IFN-γ production by LN and CNS CD4⁺ cells was measured with a mouse Th1/Th17 phenotyping kit (BD Biosciences). Briefly, stimulated cells were fixed with cold BD Cytofix™ buffer and permeabilized with BD Perm/Wash™ buffer, followed by staining with a BD Th1/Th17 phenotyping cocktail containing CD4 PerCP-Cy5.5 (clone: RM4-5), IL-17A PE (clone: TC11-18H10.1), and IFN-γ FITC (clone: XMG1.2) according to the manufacturer’s instructions. Samples were acquired using FACS Canto II flow cytometer (BD Biosciences), and the data were analyzed using Cell Quest software (Becton Dickinson, Franklin Lakes, NJ, USA).

A single-cell suspension of splenocytes was analyzed with the following anti-mouse antibodies (all from BioLegend): CD138, B220, CD80, CD19, CD44, and CD62L. Mouse Th1/Th17 Phenotyping Kit was from BD Pharmingen and performed according to the manufacturer’s instructions. Staining was typically for 30 min at 4°C. After washing, cells were fixed in 2% paraformaldehyde before analysis with a CyAn ADP Analyzer (Beckman Coulter). Further data analysis was done using FlowJo analysis software.