Development of an aequorin luminescence calcium assay for high-throughput screening using a plate reader, the LumiLux

A luminescence assay using a new plate reader, the LumiLux (PerkinElmer, Waltham, MA), has been validated for high-throughput screening (HTS). In this study, we compared the aequorin luminescence-based calcium mobilization assay to the fluorescence-based calcium assay. A cell line stably co-expressing apo-aequorin, a chimeric G-protein, and a G-protein-coupled dopamine receptor was used to screen a collection of 8,106 compounds using the Hamamatsu Photonics (Bridgewater, NJ) FDSS6000 and LumiLux as the plate readers. The assay parameters evaluated included hit rate correlation, signal-to-noise ratio, and overall assay performance calculated by Z and standard deviation. The average Z values and hit rates were comparable between assay platforms;however, the standard deviation for the agonist aequorin assay was significantly smaller. There was also a significant decrease in the number of false-positives with the aequorin assay. These results suggest that the aequorin assay in combination with the new plate reader, LumiLux, provides a simple, cost-effective, robust, and sensitive assay for HTS