Human il 8 duoset elisa kit

The concentrations of IL-8 in cell culture supernatants were measured with a sandwich enzyme-linked immunosorbent assay (ELISA) using a Human IL-8 DuoSet ELISA kit (R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s instructions. Briefly, a 96-well microplate was coated with capture antibody and incubated overnight at room temperature. Then, block plates by incubating with Block Buffer at room temperature for 1 h. The standards or cell supernatants were added to the plate; Detection Antibody, and streptavidin-HRP was added. After removal the unbound material by a washing procedure, substrate solution [1:1 mixture of color reagent A (H2O2) and color reagent B (tetramethylbenzidine)] was added in the dark. To stop the reaction, 2 N H2SO4 was added to each well. The optical density was determined at 450 nm and a reference wavelength of 570 nm using a spectrophotometer. Standard curve was constructed by plotting absorbance values versus the corresponding concentration of standard. Concentrations were calculated based on the standard curve. The limit of detection was 31.3 pg/mL.

For siRNA experiments, two days post transfection media was changed 2 hours before infection. HeLa cells were then infected with late log phase bacteria (S. Typhimurium: MOI 1; S. Typhi: MOI 30) for 60 minutes. After 60 minutes, gentamicin was added and bacteria were returned to 37°C incubator for 5 hours. Six hours post infection supernatants were collected. For overexpression experiments, media was changed 18–24 hours post infection. Six hours after the media change, supernatants were collected. Supernatants were stored at -80°C until use. Cytokine concentrations were determined using a human IL-8 DuoSet ELISA kit (R&D Systems).