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Miniaturization of Intracellular Calcium Functional Assays to 1536-Well Plate Format Using a Fluorometric Imaging Plate ReaderDepartment of Automated Biotechnology, Merck Research Laboratories, North Wales, PA.
Department of Automated Biotechnology, Merck Research Laboratories, North Wales, PA.
Department of Automated Biotechnology, Merck Research Laboratories, North Wales, PA.
Department of Automated Biotechnology, Merck Research Laboratories, North Wales, PA.
Department of Automated Biotechnology, Merck Research Laboratories, North Wales, PA. The measurement of intracellular calcium response transients in living mammalian cells is a popular functional assay for identification of agonists and antagonists to receptors or channels of pharmacological interest. In recent years, advances in fluorescence-based detection techniques and automation technologies have facilitated the adaptation of this assay to 384-well microplate format high-throughput screening (HTS) assays. However, the cost and time required performing the intracellular calcium HTS assays in the 384-well format can be prohibitive for HTS campaigns of greater than 1 x 106 wells. For these reasons, it is attractive to miniaturize intracellular calcium functional assays to the 1536-well microplate format, where assay volumes and plate throughput can be decreased by several fold. The focus of the research described in this article is the miniaturization of an intracellular calcium assay to 1536-well plate format. This was accomplished by modifying the hardware and software of a fluorometric imaging plate reader (FLIPR) to enable transfer of nanoliters of test compound directly to a 1536-well assay plate, and measure the resulting calcium response from all 1536 wells simultaneously. An intracellular calcium functional assay against the rat muscarinic acetylcholine receptor subtype 1 (rmAchR1) G-protein coupled receptor (GPCR) was miniaturized and executed on this modified instrument. In experiments measuring the activity of known muscarinic receptor agonists and antagonists, the miniaturized FLIPR assay gave EC50 and IC50 values and rank order potency comparable to the 384-well format assays. Calculated Z' factors for the miniaturized agonist and antagonist assays were, respectively, 0.56 ± 0.21 and 0.53 ± 0.22, which were slightly higher (Z'agonist = 0.55 ± 0.33) and lower (Z'antagonist = 0.70 ± 0.18) than the corresponding values in the 384-well assays. A mock agonist HTS campaign against the muscarinic receptor in miniaturized format was able to identify all wells spiked with the rmAchR1 agonist carbachol.
Key Words: GPCR (G protein-coupled receptor) • FLIPR (fluorometric imaging plate reader) • mAchR1 receptor (muscarinic acetylcholine receptor subtype 1) • HTS (high-throughput screening) • intracellular calcium • Chinese hamster ovary (CHO) • 1536-well format • homogenous assay • assay miniaturization
Journal of Biomolecular Screening, Vol. 9, No. 5,
417-426 (2004) This article has been cited by other articles:
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