Elisa kits for il 2

Lymphocyte cultures were tested for reactivity in cytokine release assays using commercially available ELISA kits for IL-2, IFNγ, and TNFα (R&D Systems, Minneapolis, MN, USA). For these assays, 1 × 10⁵ responder cells (T-cells) and 1 × 10⁵ stimulator cells (tumor cells) were incubated in a 0.2-ml culture volume in individual wells of 96-well plates. Stimulator cells and responder cells were cocultured for 18 h. Cytokine secretion was measured in culture supernatants diluted to be in the linear range of the assay. As a control for T cell activity, we incubated the different T-cell cultures with PMA/ionomycin at a concentration of 50 ng/ml and 1 µM, respectively.

Lymphocyte cultures were tested for reactivity in cytokine release assays using commercially available ELISA kits for IL-2, IFNγ and TNFα(R&D Systems, Minneapolis, MN). For these assays, 1×10⁵ responder cells (T-cells) and 1×10⁵ stimulator cells (tumor cells) were incubated in a 0.2-ml culture volume in individual wells of 96-well plates. Stimulator cells and responder cells were co-cultured for 18 h. Cytokine secretion was measured in culture supernatants diluted to be in the linear range of the assay. As a control for T cell activity, we incubated the different T-cell cultures with PMA/Ionomycin at a concentration of 50ng/ml and 1μM respectively.

Ethyl pyruvate and Con A were purchased from Sigma-Aldrich (USA). Enzyme-linked immunosorbent assay (ELISA) kits for IL-2, IL-6, TNF-α and IL-1β were purchased from R&D systems (Minneapolis, MN, USA). HMGB1 ELISA kit was purchased from Shino-Test Corporation (Oonodai, Kanagawa, Japan). Antibodies used in this study include HMGB1 (Epitomics, CA), IL-2 (Biolegend, CA), IL-6 (Proteintech, CA), TNF-α (Santa Cruz, CA), NF-κB (Proteintech, CA), IL-1β (Biolegend, CA), IκB α (Cell signal technology, CA) and IκB β (Cell signal technology, CA).