Cancer cells were seeded in 96 well round-bottom plates (1 x 105 cells/well) overnight, before addition of untransduced T cells or CAR T cells (1 x 105 cells/well). Alternatively, the anti-idiotypic monoclonal antibody BW2064/36 was coated overnight on 96 well plates at the indicated concentrations before adding CAR T cells (1 x 105 cells/well). After 48 hours of co-culture, IL-2 and IFN-γ in culture supernatants were detected by ELISA as previously described (33 (link)). TNF-α was detected using the “human TNF-α Standard ABTS ELISA Development Kit” (PeproTech, Cranbury, NJ, USA) according to the manufacturer´s instructions.
Human tnf α standard abts elisa development kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Human TNF-α Standard ABTS ELISA Development Kit is a laboratory reagent designed for the quantitative measurement of human tumor necrosis factor alpha (TNF-α) in biological samples. The kit utilizes the enzyme-linked immunosorbent assay (ELISA) technique and ABTS as the chromogenic substrate.
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3 protocols using human tnf α standard abts elisa development kit
1
CAR T Cell Cytokine Release Assay
2
Cytokine Measurement in Plasma
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The levels of IL-6 and IL-10 were measured in plasma using the immunoenzymatic method called enzyme-linked immunosorbent assay (ELISA) (BD Bioscience, San Diego, CA, USA) in accordance with the manufacturer's instructions. Readings were performed using a Multiskan microplate spectrophotometer (Thermo LabSystems, Milford, MA, USA) at a 450 nm wavelength. The range of detection of the IL-6 kit used was 4.68 to 300 pg/mL, while the range of the IL-10 kit was from 7.8 to 500 pg/mL.
The TNF-α cytokine was measured using the Human TNF-α Standard ABTS ELISA Development Kit (Peprotech, Cranbury, NJ, USA) in accordance with the manufacturer's instructions. After 25 minutes, optical density was read using the Multiskan microplate spectrophotometer (Thermo Labsystems, USA) at 405 nm, using the 620 nm filter as reference wavelength. The detection range of the test was 62.5 to 4,000 pg/mL.
The TNF-α cytokine was measured using the Human TNF-α Standard ABTS ELISA Development Kit (Peprotech, Cranbury, NJ, USA) in accordance with the manufacturer's instructions. After 25 minutes, optical density was read using the Multiskan microplate spectrophotometer (Thermo Labsystems, USA) at 405 nm, using the 620 nm filter as reference wavelength. The detection range of the test was 62.5 to 4,000 pg/mL.
3
Quantifying Plasma MIF and TNF-α
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We detected the macrophage inhibitor factor (MIF) from plasma samples using MIF Human ELISA Kit (ThermoFisher™). We also analyzed tumor necrosis factor alpha (TNF-α) by Human TNF-α Standard ABTS Elisa Development Kit (Peprotech). Both kits were used according to the manufacturer instructions.
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