Human il 2 elisa kit

Human peripheral blood mononuclear cells (PBMC), isolated from healthy donor leukocones (NHSBT) using Ficoll-Paque Plus (GE Healthcare), were cultured at 2 × 10⁵ cells/well in 96-well plates (Corning) pre-coated with 0.5 μg/mL anti-human CD3 monoclonal antibody (OKT3, eBioscience). Cultures were supplemented with 100 ng/mL staphylococcal enterotoxin A (SEA) (Sigma), 30 μg/mL tremelimumab or isotype control (MedImmune) and selumetinib at the concentrations indicated. Following 72 h incubation at 37 °C, IL-2 release was measured by DELFIA using reagents from a human IL-2 ELISA kit (R&D systems) and europium-labelled streptavidin (Perkin-Elmer). Results were repeated in two independent experiments.

Plasma cytokine concentration was assessed using the following ELISA kits (as recommended by the kit manufacturer): Human IFN-γ ELISA Kit, sensitivity < 8 pg/mL (R&D Systems, Minneapolis, MN, USA), Human IL-2 ELISA Kit, sensitivity < 7 pg/mL (R&D Systems, USA), Human IL-4 HS ELISA Kit, sensitivity 0.03–0.22 pg/mL (R&D Systems, USA) and Human IL-10 HS ELISA Kit, sensitivity 0.03–0.17 pg/mL (R&D Systems, USA).
The light absorbance readings in the tested samples were performed using an automated VICTOR reader (Perkin Elmer, Waltham, MA, USA). The concentration of the samples was assessed on the basis of a standard curve made of standards with different concentrations and plotted by the WorkOut computer program.

Anti-phosphorylated mTOR (S2448) antibodies and anti-Bcl2 were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Anti-p65, anti-PARP, anti-IκBα, anti-phosphorylated IκBα (S32), anti-β-actin, anti-phosphorylated ERK (T202/Y204), anti-ERK, anti-phosphorylated p38 (T180/Y182), anti-p38, anti-phosphorylated JNK (T183/Y185), anti-JNK, anti-phosphorylated c-Jun (S63), and anti-c-Jun were obtained from Cell Signaling Technology (Danvers, MA, USA). MTT (1-(4,5-Dimethylthiazol-2-yl)-3,5-diphenylformazan), phorbol 12-myristate 13-acetate (PMA), A23187, and dinitrochlorbenzene (DNCB) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Anti-human CD3 and CD28 antibodies were obtained from Bioxcell (West Lebanon, NH, USA). Human IL-2 ELISA kit was purchased from R&D systems (Minneapolis, MN, USA). AnnexinV staining dye and caspase3/7 staining dye for IncuCyte^® cell imaging system were obtained from Essen bio (Ann Arbor, MI, USA). Antibodies to CD69, CD25, and CD40L were purchased from ebiosciences (San Diego, CA, USA). NE-PER Nuclear and Cytoplasmic Extraction Reagents Kit was obtained from Thermo Fisher Scientific (Waltham, MA, USA). Mouse IgE ELISA kit was purchased from BD Biosciences (San Diego, CA, USA). House dust mite extract was obtained from Greer (Lenoir, NC, USA).