Adaptation of aequorin functional assay to high throughput screening.
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.
Le Poul
Emmanuel
E
Euroscreen s.a., Brussels, Belgium.
Hisada
Sunao
S
Mizuguchi
Yoshinori
Y
Dupriez
Vincent J
VJ
Burgeon
Emmanuel
E
Detheux
Michel
M
D000331:Aequorin / Q000032:analysis* / Q000737:chemistry*; D000818:Animals; D001331:Automation; D001709:Biotechnology / Q000295:instrumentation / Q000379:methods*; D016466:CHO Cells; D006224:Cricetinae; D004305:Dose-Response Relationship, Drug; D006801:Humans; D008024:Ligands; D064446:Orexin Receptors; D017785:Photons; D044403:Receptor, Serotonin, 5-HT2B; D043562:Receptors, G-Protein-Coupled; D018013:Receptors, Neuropeptide / Q000032:analysis; D011979:Receptors, Pituitary Hormone / Q000032:analysis; D011985:Receptors, Serotonin / Q000032:analysis; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence / Q000295:instrumentation / Q000379:methods*; D013997:Time Factors
D008024:Ligands; D064446:Orexin Receptors; D044403:Receptor, Serotonin, 5-HT2B; D043562:Receptors, G-Protein-Coupled; D018013:Receptors, Neuropeptide; D011979:Receptors, Pituitary Hormone; D011985:Receptors, Serotonin; C092274:melanin-concentrating hormone receptor; D000331:Aequorin