Alphalisas

Primary hMDM cultured at 1 x 10⁵ cells per cm² in 48-well plates (BD Falcon) were incubated for 24 hours with dopamine (10⁻⁶ M) or the positive control LPS (1 ng/ml) prior to the collection of supernatants and lysates to examine IL-1β production. At 30 min prior to this collection, some plates were stimulated with ATP (2.5 mM) to induce inflammasome activation, and supernatants were collected for the analysis of active caspase-1 and IL-1β secretion. For NF-κB inhibition assays, cells were pretreated for 45 minutes with BAY 11–7082 (10⁻⁵M) prior to dopamine or LPS treatment. After 24 hr incubation with dopamine or LPS in the presence of BAY 11–7082, supernatants and lysates were collected for the analysis of IL-1β production. Cytokine concentration was quantified via AlphaLISAs performed according to the manufacturer’s protocol (PerkinElmer). The limit of detection was 0.6 pg/mL for IL-1β for AlphaLISA. Caspase-1 concentration in supernatants was determined via Quantikine^® ELISA performed according to the manufacturer’s protocol, and the limit of detection for this assay was 0.68 pg/mL (R&D systems, Minneapolis, MN).