Mouse il 2 quantikine elisa kit

The I-A^b restricted Eα-specific 1H3.1 T-cell hybridoma cell line was used for activation assays to recognize preactivated dendritic cells expressing I-A^b::Eα^52–68 complexes. T cells were cultured in RPMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum (Wisent) and 55 μM β-mercaptoethanol at 37°C with 5% CO₂. For activation assays, preactivated DCs were mildly fixed in 1% PFA for 15 minutes at room temperature. Following fixation, cells were washed with PBS 3 times and then quenched thrice in complete medium for 10 minutes each at room temperature. After, 1H3.1 T cells and fixed DCs were co-cultured at 2:1 and incubated for 40 h at 37°C with 5% CO₂. Next, the tissue culture medium was collected, and T cells were immediately isolated following centrifugation at 800g for 5 minutes. The supernatant was immediately stored in −80°C for downstream IL-2 secretion analysis. To quantify T-cell activation, secreted IL-2 samples were diluted 1:10 and subsequently analysed using Mouse IL-2 Quantikine ELISA Kit (R&D Systems Inc, Minneapolis, MN) as per manufacturer’s specifications.