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Adaptation of Aequorin Functional Assay to High Throughput Screening

Euroscreen s.a., Brussels, Belgium

Sunao Hisada

Hamamatsu Photonics K.K., Systems Division, Hamamatsu City, Japan

Yoshinori Mizuguchi

Hamamatsu Photonics K.K., Systems Division, Hamamatsu City, Japan

Vincent J. Dupriez

Euroscreen s.a., Brussels, Belgium

Emmanuel Burgeon

Euroscreen s.a., Brussels, Belgium

Michel Detheux

Euroscreen s.a., Brussels, Belgium

AequoScreen^TM, a cellular aequorin-based functional assay, has been optimized for luminescent high-throughput screening (HTS) of G protein-coupled receptor (GPCRs). AequoScreen is a homogeneous assay in which the cells are loaded with the apoaequorin cofactor coelenterazine, diluted in assay buffer, and injected into plates containing the samples to be tested. A flash of light is emitted following the calcium increase resulting from the activation of the GPCR by the sample. Here we have validated a new plate reader, the Hamamatsu Photonics FDSS6000, for HTS in 96- and 384-well plates with CHO-K1 cells stably coexpressing mitochondrial apoaequorin and different GPCRs (AequoScreen cell lines). The acquisition time, plate type, and cell number per well have been optimized to obtain concentration-response curves with 4000 cells/well in 384-well plates and a high signal: background ratio. The FDSS6000 and AequoScreen cell lines allow reading of twenty 96- or 384-well plates in 1 h with Z’ values of 0.71 and 0.78, respectively. These results bring new insights to functional assays, and therefore reinforce the interest in aequorin-based assays in a HTS environment.

Journal of Biomolecular Screening, Vol. 7, No. 1, 57-65 (2002)
DOI: 10.1177/108705710200700108


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