Human tnf alpha simplestep elisa kit

2 × 10⁵ 16HBE cells were seeded into cell culture dishes and transfected with corresponding shRNA at 37 °C for 24 h. After 1 h treatment with or without 30 μM asiatic acid, an agonist of p38 MAPK at 37 °C, 8 ml fresh RPMI-1640 completement medium containing 2% CSE were replaced and cultured for additional 48 h. Subsequently, Human TNF alpha SimpleStep ELISA® Kit, Human IL-1 beta SimpleStep ELISA® Kit, Human IL-6 SimpleStep ELISA® Kit (abcam) were used to determine the levels of inflammatory cytokines in culture supernatant according to the manufacturer’s commendation. The normal 16HBE cells without transfection and treatment were used as control.

The TNF-α release in cell culture supernatant was quantified by human TNF alpha SimpleStep ELISA^® Kit (ABCAM, AB181421), according to the manufacturer’s protocol. Briefly, after 1 h of treatment, cell culture supernatants were collected. Volumes of 50 μL of samples or standard were loaded to appropriate wells. After which, 50 μL of the antibody cocktail were added to each well. The plate was incubated for 1 h at room temperature on a plate shaker set to 400 rpm. The plate was then washed 3 times with 50 μL 1X Wash Buffer PT in each well. Then, 100 μL of TMB development solution were added to each well and incubated for 10 min in the dark on a plate shaker set to 400 rpm. Finally, 100 μL of stop solution in each well were added; the absorbance was read at 450 nm. TNF-α (pg/μL) concentration was calculated by extrapolation of the data in a standard curve constructed with known concentrations of TNF-α.
To obtain more accurate data and to exclude misleading results due to eventual differences in the amount of proteins released in the supernatant, data from TNF-α ELISA were normalized by µg of total proteins in each well. Total protein was calculated by bicinchoninic acid (BCA) assay using a BSA standard curve dilution.

TNF-α concentrations in undiluted or appropriately diluted supernatants were determined by ELISA using the SimpleStep human TNF alpha ELISA kit (Abcam, Cambridge, MA), performed following the manufacturer’s instructions and with endpoint absorbance measurements at 450 nm on an Infinite M200 Pro plate reader (Tecan, Männedorf, Switzerland). For each experiment, absorbance measurements from wells containing a twofold dilution series of purified TNF-α (31.25 pg/mL to 2000 pg/mL, in duplicate) were used to calculate a calibration curve using a four-parameter logistic fit, which in turn was used to calculate TNF-α concentrations in all sample wells. Concentrations of CXCL10 and IL-10 were determined equivalently using SimpleStep human IP-10 ELISA kit (Abcam) and the SimpleStep human IL-10 ELISA kit (Abcam), respectively, following the manufacturer’s instructions. When handling multiple 96-well plates simultaneously, plates were staggered in 3-min intervals starting with the last wash step to ensure that incubation times with the development solution and stop solution were constant.