Quantifying TNFα Production in SHSY-5Y Cells Under Oxidative Stress

Following the manufacturer’s instruction, TNFα production on the SHSY-5Y cell line under oxidative stress conditions was analyzed by the Human Tumor Necrosis Factor α ELISA Kit (Merck Life Science, Rome, Italy). Next, 100 µL of material was placed in each well of a 96-well ELISA plate, incubated at room temperature for 2 h, and overnight at 4 °C. Five washings with washing buffer followed incubation, and adding 100 μL of biotinylated anti-TNFα to each well. After a 2-h room temperature incubation, each well was aspirated and cleaned five times before adding 100 μL Streptavidin–HRP for a 1-h incubation. Incubate 100μL of chromogen solution in each well for 30 min at room temperature in darkness after washing the plate with Streptavidin–HRP solution. The absorbance of each well was measured at 450 nm using a Tecan plate reader after applying Stop Solution [41 (link)]. The data are presented as mean SD (%) relative to the untreated control sample (0% line).