IL-1β Secretion Assay

Levels of secreted IL-1β were measured using the BD OptEIA™ Human IL-1β enzyme-linked immunosorbent assay (ELISA) kit (BD-Pharmingen, San Diego, CA). This kit is highly selective for IL-1β and does not cross react with other proteins including other cytokines. A 96-well micro well plate, designed for ELISA assays (Fisher, Pittsburgh, PA), was coated with a capture antibody for IL-1β that was diluted in coating buffer. The ELISA plate was incubated with the capture antibody overnight at 4 ˚C. After the incubation, the capture antibody was removed and blocking solution (PBS and bovine calf serum) was added to each well and incubated at room temperature for 1h. Following the blocking step, cell supernatants and IL-1β standards were added to the plate and incubated for 2 h at room temperature. Detection antibody linked to horseradish peroxidase (HRP) was then added followed by substrate. The reaction was stopped by addition of 1 M phosphoric acid and absorbance was measured at 450 nm on a Thermo Labsystems Multiskan MCC/340 plate reader (Fisher Scientific).