Human il 2 elisa kit

IL-2 cytokine released into cell culture supernatant was quantified using human IL-2 ELISA kit (BD Biosciences) according to the manufacturer’s instructions. Jurkat cells were stimulated with anti-CD3 (5 μg/ml) for the indicated times prior to addition of cell lysis buffer (Cell Signaling). Following PMA/Ionomycin stimulation for 15 minutes, nuclear proteins were isolated using nuclear protein isolation kit (NEPER, Thermo Scientific) following manufacturer’s instructions. Proteins were separated by polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes (BIORAD). Membranes were incubated in protein-free blocking buffer (Thermo Scientific) for 1 hour at room temperature followed by incubation with primary antibodies. Proteins were detected with Amersham ECL (GE Healthcare) using a Kodak Imager. Primary antibodies used were: NFAT and pLAT(Y226; BD Biosciences); total LAT (Biolegend); pZAP70 (Y319); total ZAP70; pLck (Y394/ pSrcY416); total Lck (Y394); Csk; YY1 (all from Cell Signaling Technology); PTPRE (Origene, clone 4B2). Immunoblots were quantified by ImageJ.

The transfected cells and sorted γδT cells were separately incubated with 10 ng/mL NSP8 protein (Sino Biological Inc., Beijing, China) or control protein for 30 min at room temperature. After extensive washing with RPMI-1640 culture medium, the transfected cells and natural γδT cells were then plated into 24-well plates at 1 × 10⁶ cells per well. The supernatants were harvested after 24 h and the level of IL-2 was detected by using Human IL-2 ELISA Kit (BD Biosciences, San Jose, CA, United States) according to the manufacture’s instructions.