Human tumor necrosis factor α elisa kit

According to the guidelines provided by the manufacturer, an analysis of TNFα production on the SHSY-5Y supernatant under conditions of oxidative stress was conducted utilizing the Human Tumor Necrosis Factor α ELISA Kit (Merck Life Science, Milan, Italy). Subsequently, 100 µL of the sample was dispensed into individual wells of a 96-well ELISA plate, followed by an incubation period at room temperature lasting 2 h, then overnight at 4 °C. This was succeeded by five consecutive washes using a washing buffer post-incubation and adding 100 µL of biotinylated anti-TNFα into each well. Following a 2 h incubation at room temperature, the contents of each well were aspirated and washed five times before the introduction of 100 µL Streptavidin–HRP for a 1 h incubation. Subsequently, the plate with Streptavidin–HRP solution, 100 µL of chromogen solution, was incubated in each well for 30 min at room temperature without light. The absorbance of each well was then determined at 450 nm utilizing a Tecan plate reader after applying stop solution [67 (link)]. The data are presented as mean ± SD (%) relative to the untreated control sample (0% line).

TNFα production on SHSY-5Y cells under oxidative stress was analyzed by the Human Tumor Necrosis Factor α ELISA Kit (Merck Life Science, Rome, Italy) following the manufacturer’s instructions. Briefly, 100 µL of SHSY-5Y’s lysate was added to each well of a 96-well ELISA plate, and the plate was incubated at room temperature for 2 h, followed by overnight incubation at 4 °C. At the end of incubation, wells were washed five times with a washing buffer, and 100 μL of biotinylated anti-TNFα was added to each well. After 2 h of incubation at room temperature, the solution in each well was aspirated, the wells were washed five times and 100 μL of streptavidin-HRP was added to each well and incubated at room temperature for 1 h. After washing, 100 μL of chromogen solution was added to each well and incubated for 30 min at room temperature and in the dark. The absorbance of each well was measured after the addition of stop solution at 450 nm using a plate reader (Infinite 200 Pro MPlex, Tecan, Männedorf, Switzerland) [50 (link)].

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).