Adaptation of Aequorin Functional Assay to High Throughput Screening

Abstract

AequoScreen, 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.

Corazonin receptor signaling in ecdysis initiation

“…Ligand-mediated activation of the receptor produced elevation of intracellular Ca 2ϩ and luminescence responses via activation of an aequorin-coelentrazine complex (20,23). In CHO cells, MasCRZR had the same ligand selectivity as in Xenopus oocytes; no other peptides including 100 nM EH evoked responses.…”

Phylogenetic Diversity of Sponge-Associated Fungi from the Caribbean and the Pacific of Panama and Their In Vitro Effect on Angiotensin and Endothelin Receptors

“…Detection of the calcium signal is based on the interaction of calcium ions with the calciumbinding bioluminescent complex aequorin. Upon calcium binding, aequorin oxidizes coelenterazine-h into coelenteramide with production of CO 2 and emission of light (466 nm) (Le Poul et al 2002). Confluent CHO-AEQ cells that were grown and transfected in 75-cm 2 culture flasks were harvested by a brief treatment with Trypsin/EDTA solution, centrifuged, and resuspended in DMEM-F12 cell culture medium (Life Technologies Europe B.V., Gent, Belgium) containing 0.1 % BSA (Sigma, Bornem, Belgium) and denoted as assay buffer, at a cell density of 2.5×10 6 cells/ml.…”

Vasopressin-like peptide and its receptor function in an indirect diuretic signaling pathway in the red flour beetle

“…Transfection was performed using FuGene6 (Roche) according to the manufacturer's protocol at a ratio of DNA to FuGene6 of 3:1. Cell suspensions were loaded with coelenterazine 3 h (Invitrogen) before testing according to a previously developed protocol (Park et al, 2003;Poul et al, 2002).…”