Baf210

Measurement of secreted TNFα in the supernatants was determined by enzyme-linked immuno sorbent assay (ELISA). Briefly, 0.1 mL of 4 mg/mL monoclonal anti-human TNFα capture antibody (MAB610, R&D Systems) was added to 96-well plates for overnight incubation at room temperature. Wells were washed with PBS (HyClone) containing 0.05% Tween-20 and blocked with 0.3 mL PBS containing 1% bull serum albumin (BSA), 5% sucrose and 0.05% NaN₃ for 1 h at room temperature. After washing, successive additions of 0.05 mL samples or standards (2 h), 0.1 mL biotinylated polyclonal anti-human TNFα detection antibody (BAF210, R&D Systems) in 20 mmol/L Tris with 150 mmol/L NaCl and 0.1% BSA (2 h), 0.1 mL streptavidin-HRP (R&D Systems) diluted 200 times with PBS containing 1% BSA (20 min), and 0.1 mL of equal volumes of 3,3',5,5'-tetramethylbenzidine and hydrogen peroxide (KPL, Gaithersburg, MD, United States) (30 min). The reaction was stopped by the addition of 1% H₂SO₄ solution. The optical density of each sample was analyzed at 450 nm with a reference reading at 630 nm using a SpectraMax 340 absorbance platereader (Molecular Devices, Union City, CA, United States). A standard curve was constructed by sequential dilution of a TNFα standard from 2000-15 pg/mL. The concentration of TNFα in the experimental samples was calculated from the standard curve