Human tnf alpha uncoated elisa

To investigate the functionality of NK92 cells encapsulated in a porous hydrogel, crosslinked alginate in the hydrogel was degraded using 8 mM Ethylenediaminetetraaceticacid (EDTA) (Supplementary Fig. 3) [29 (link)]. The hydrogel was incubated for 30 min and centrifuged to obtain 3D cultured cells. The cytotoxicity was investigated using a calcein-AM-based assay. Target cancer cells were stained with calcein-AM (Invitrogen, USA) for 1 h at 37 °C. The labeled target cells (1 × 10⁴ cells / 100 µL) and NK92 cells were placed in 96-well round-bottom plates at effector-to-target (E/T) ratios of 2:1 and 1:1 for 4 h. The maximum release was measured by adding 4% Triton X-100 to the target cell. The percentage cytotoxicity was calculated using the following formula: (sample release – spontaneous release) / (maximum release – spontaneous release) × 100.
Cytokine release, which affects the target, was measured using an ELISA. The supernatant of the tumor cell media co-cultured with NK cells was collected, and the concentration of released cytokines (interferon gamma, TNF-alpha) was investigated. An ELISA kit (Human IFN gamma uncoated ELISA, Human TNF alpha uncoated ELISA; Invitrogen, USA) was used according to the instructions of the manufacturer.

Freshly harvested iPS-MΦ were plated at 3 × 10⁴ cells per well in a 96-well plate in 100 µL macrophage media and treated with 10 ng/mL LPS (Sigma), 10 µM zVAD-fmk (BD Pharmingen^TM-550377) and/or 3 µM GSK872 (BioVision-2673-5). Supernatant was collected after 3 h and stored at −80 °C before ELISA. Human TNF alpha Uncoated ELISA (Invitrogen-88-7346-22) was performed according to manufacturer’s instructions, media samples were diluted 1:50 and 100 µL was used.

Conditioned medium from activated murine splenocytes were harvested as described and processed for enzyme-linked immunosorbent assay (ELISA). ELISA for TNFα (Mouse TNFalpha ELISA ReadySet-Go! 10x #88-7324-86, eBioscience), IFNγ (Mouse IFN gamma ELISA Ready-SET-Go! 10x #88-7314-86, eBioscience), and IL-10 (Mouse IL-10 ELISA Ready-SET-Go! #88-7105-86, eBioscience) was performed according to the manufacturer's instructions in triplicates and optical density was measured with a microplate reader Tecan Infinite (Tecan, Männedorf, Switzerland). A standard curve was generated with a four parametric logistic curve fit.
Conditioned medium from activated human PBMC were harvested as described and processed for quantitative cytokine detection via ELISA. ELISA for human TNFα (Human TNF alpha Uncoated ELISA, 88-7346, Invitrogen) and human IFNγ (Human IFN gamma Uncoated ELISA, 88-7316, Invitrogen) was performed according to the manufacturer's instructions in triplicates and optical density was measured with a microplate reader Tecan Infinite (Tecan, Männedorf, Switzerland). A standard curve was generated with a four parametric logistic curve fit.