Miniaturization of intracellular calcium functional assays to 1536-well plate format using a fluorometric imaging plate reader
| Autor: | Peter Hodder, Jason Cassaday, Kurtis Berry, Berta Strulovici, Rebecca Mull |
|---|---|
| Rok vydání: | 2004 |
| Předmět: |
0301 basic medicine
Agonist Carbachol medicine.drug_class chemistry.chemical_element Calcium 01 natural sciences Biochemistry Calcium in biology Fluorescence Analytical Chemistry 03 medical and health sciences GTP-Binding Proteins Muscarinic acetylcholine receptor medicine Animals Receptor Calcium metabolism Miniaturization Chemistry Molecular biology Receptors Muscarinic 0104 chemical sciences Rats 010404 medicinal & biomolecular chemistry 030104 developmental biology Biophysics Molecular Medicine Plate reader Biotechnology medicine.drug |
| Zdroj: | Journal of biomolecular screening. 9(5) |
| ISSN: | 1087-0571 |
| Popis: | 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 10(6) 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 EC(50) and IC(50) 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. |
| Databáze: | OpenAIRE |
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