Thermo labsystems multiskan mcc 340 plate reader

TNFα levels were measured using the BD OptEIA^TM Human TNFα enzyme-linked immunosorbent assay (ELISA) kit (BD-Pharmingen, San Diego, CA). Briefly, a 96-well micro well plate, designed for ELISA (Fisher, Pittsburgh, PA), was coated with a capture antibody for TNFα diluted in coating buffer. The plate was incubated with the capture antibody overnight at 4° C. After incubation, the capture antibody was removed by washing the plate three times with wash buffer (PBS and 0.05% Tween-20). Assay diluent (PBS and bovine calf serum) was added to each well (blocking non-specific binding) and incubated at room temperature for 1h. The assay diluent was removed by washing the plate three times, and the cell supernatants and TNFα standards were added to the coated plated and incubated for 2h at room temperature. Following this incubation, the plate was thoroughly washed five times and then incubated for 1h with a detection antibody linked to horseradish peroxidase (HRP). The detection antibody-HRP complex was removed by washing the plate seven times and then the plate was incubated for 30 min with substrates for horseradish peroxidase. The incubation with the substrates was ended by addition of acid and the absorbance was measured at 450 nm on a Thermo Labsystems Multiskan MCC/340 plate reader (Fisher Scientific).

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).

IFNγ and TNFα levels were assessed using the OptEIA™ enzyme-linked immunosorbent assay (ELISA) human IFNγ and TNFα kits (BD-Pharmingen, San Diego, CA) respectively. Briefly, appropriate capture antibody was applied to the wells of a 96 well flat-bottom microwell plate specifically designed for ELISA (Fisher, St. Louis MO) after removal of excess capture antibody (by washing with PBS containing 0.05% Tween-20), the wells were treated with blocking buffer. Blocking buffer was removed and cell supernatants and IFNγ or TNFα standards were added to the plate. Following the incubation with samples and standards, detection antibody was added. Following the removal of the detection antibody, a substrate solution was added to each well. Incubation with substrate was ended by addition of acid and the absorbance measured at 450 nm on a Thermo Labsystems Multiskan MCC/340 plate reader (Fisher Scientific).

IL-1β levels were measured using the BD OptEIA^™ Human IL-1β enzyme-linked immunosorbent assay (ELISA) kit (BD-Pharmingen, San Diego, CA). Briefly, a 96-well micro well plate, designed for ELISA (Fisher, Pittsburgh, PA), was coated with a capture antibody for IL-1β diluted in coating buffer. The plate was incubated with the capture antibody overnight at 4 °C. After incubation, the capture antibody was removed by washing the plate three times with wash buffer (PBS and 0.05% Tween-20). Assay diluent (PBS and bovine calf serum) was added to each well (blocking non-specific binding) and incubated at room temperature for 1h. The assay diluent was removed by washing the plate three times, and the cell supernatants and IL-1β standards were added to the coated plated and incubated for 2 h at room temperature. Following this incubation, the plate was thoroughly washed five times and then incubated for 1h with a detection antibody to IL-1β which was conjugated with horseradish peroxidase. The excess detection antibody was removed by washing seven times and a substrate solution was added for 30 min at room temperature to produce a colored product. The incubation with the substrate was ended by addition of acid and the absorbance was measured at 450 nm on a Thermo Labsystems Multiskan MCC/340 plate reader (Fisher Scientific).