Opteia human tnf elisa kit

After 22 h-incubation of 5×10³ PC3-PSCA tumor cells with 5×10⁴ freshly isolated PBMCs or isolated CD8+ T cells in the presence or absence of 30 pmol/ml of recombinant Abs, cells were spun down and supernatants were collected. T cells of one triplet were pooled, stained with anti-CD25/PE, anti-CD69/PE-Cy5, and anti-CD8/FITC (Miltenyi Biotec GmbH) and measured using a flow cytometer. Cytokine concentrations in collected supernatants were determined using OptEIA Human IFN-γ and OptEIA Human TNF ELISA Kits (BD Biosciences).

For activation experiments, 1×10⁵ gene modified T cells were seeded in 96-well plates in triplets together with target cells. TMs were added at the indicated concentrations. After a 48h- cultivation CD25 surface expression on T cells was analyzed using a MACSQuant Analyzer®. Cell free supernatants were harvested after 24h from cultures to determine cytokine concentrations by using OptEIA™ Human IFN-γ, OptEIA™ Human IL-2, OptEIA™ Human IL-6, and OptEIA™ Human TNF ELISA Kits (BD Biosciences, Heidelberg, Germany). Alternatively, cell culture supernatants were analyzed using the MACSPlex Cytokine 12 Kit (Miltenyi Biotec GmbH), a MACSQuant^® Analyzer (Miltenyi Biotec GmbH) and the MACSQuantify^® software (Miltenyi Biotec GmbH) according to the manufacturer‘s instructions.

To compare different activation stimuli 1*10⁵ modified or control T cells were seeded in 96-well plates in triplets and either Emab coated beads, αCD3/CD28 coated polyclonal activator beads or PSCA expressing PC3 cells were added to T cell cultures at a 1∶4 ratio. Absolute cell numbers of triplets were quantified at day 0 and at indicated time points. To determine cytokine release, cells were spun down and supernatants were collected. The cytokine concentration in the supernatants was determined by using the OptEIA™ Human IFN-γ, OptEIA™ Human IL-2 and OptEIA™ Human TNF ELISA Kits (BD Biosciences, Heidelberg, Germany). Experiments with expanded and purified ECAR T cells to determine cytokine release in the presence of tumor target cells were performed in the same way. For expansion experiments with expanded and purified ECAR T cells similar numbers of T cells and eFluor670 (eBioscience, San Diego, USA) stained target cells were seeded in 96-well plates in triplets in culture medium without the addition of exogenous cytokines and absolute cell numbers of triplets were quantified at day 0 and indicated time points from an aliquot (at least 1/10 of total sample volume) using a MACSQuant Analyzer.