Anti cd3 ab

Primary B cells isolated from the spleen/LN (sorted for CD19⁺ or B220⁺) were stimulated with LPS (1 µg/ml). CD4⁺ T cells (>98%) from the spleen and/or LN were activated in plate-bound anti-CD3 Abs (10 µg/ml) and soluble anti-CD28 Abs (1 µg/ml) as recommended by the manufacturer (BD Pharmingen, San Diego, CA, USA) and as previously described^{25 (link)}. For intracellular cytokine detection, cells were re-stimulated for 5 h with PMA (Sigma, P8139) (20 ng/ml) and ionomycin (Sigma I0634) (1 µM). Golgi-stop was added in the last hour and intracellular cytokine staining was performed using BD Biosciences Cytofix/Cytoperm kit as recommended (BD Pharmingen, San Diego, CA, USA). FACS analysis was performed on a Becton-Dickinson FACSCalibur (BD Biosciences) using protein-specific monoclonal antibodies and corresponding isotype control Abs (PharMingen, San Diego, CA, USA) as previously described^{13 (link), 25 (link)}. FACS analysis was performed on samples stained with mAbs conjugated with fluorescent dyes and each experiment was color-compensated. Dead cells were stained with dead cell exclusion dye (Fixable Viability Dye eFluor 450; eBioscience) and live cells were subjected to side-scatter (SSC) and forward scatter (FSC) analysis. Quadrant gates were set using isotype controls with less than 0.2% background.

Human cord blood was obtained from informed consented mothers and peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation through Lymphoprep (Nycomed). CD4⁺ T cells from human PBMC and murine spleen were purified by negative selection (AutoMACS; MiltenyiBiotec). T cells were cultured in RPMI 1640 supplemented with 10% FCS, 2 mM l-glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin. Purified CD4⁺ T cells (purity ≥98%, 2 × 10⁶ cells/ml) were activated with plate-bound anti-CD3 Abs (3 μg/ml; BD Biosciences), rIL-12, and different doses of rIL-33 or a combination of these cytokines for different times as indicated. Freshly isolated cells, DLNs or spleen of immunized mice were cultured with different dose of OVA peptide without cytokines for 1–3 days. The cells, cellular RNA and culture supernatants were collected for analysis by flow cytometry, PCR or ELISA, respectively.

Caspase activity was determined using CellEvent Caspase-3/7 Green Flow Cytometry Assay Kit (Invitrogen), as per the manufacturer’s instruction. Mature T cells purified from the spleen and lymph nodes (10⁵) were seeded in 100 μL of complete RPMI into 96-well round bottom plates coated with anti-CD3 abs (1 μg/ml; BD Pharmingen 553058) for 2 h. Anti-CD28 abs (0.2 μg/ml; BD BioSciences 553294) were added into each well. After incubation at 37°C for the indicated time, CellEvent Caspase-3/7 Green Detection Reagent (400 nM) was then added into T cell suspensions for 30 min at 37°C and analyzed using an LSR II flow cytometer.