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Quencher Dyes Can Alter the Pharmacokinetic Response Profiles of Living Cells in Fluorescence-based Calcium Mobilization AssaysTélécharger
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April 21, 2010
Authors: Dee Shen, Wayne F. Patton, Enzo Life Sciences, Farmingdale, NY ; Paul Held, Peter Banks, BioTek Instruments, Winooski, VT
G protein-coupled receptors (GPCRs) have been the target of choice for numerous high-throughput screening (HTS) campaigns designed to search for new drug leads of potential therapeutic signifi cance. By coupling receptors to Gq proteins, which stimulate intracellular calcium fl ux upon agonist binding, a functional response can readily be measured using various calcium-sensitive dyes and a fl uorescence microplate reader. GPCR assays using fl uorescent dyes, such as Fluo-3 AM or Fluo-4 AM, typically require both a serum-containing medium removal step and then a wash step after dye loading to reduce background fl uorescence signal. These wash steps may introduce assay variation, especially when loosely adherent cells become dislodged. Consequently, dyes have been introduced for the purpose of quenching extracellular background fl uorescence in calcium fl ux assays. These include trypan blue, brilliant black, hemoglobin and proprietary dyes available in certain commercial no-wash calcium assay kits (C. Mehlin, et al.; Biotechniques 34, 164 (2003); D.G. Cronshaw, et al.; J Leukoc. Biol. 79, 1369 (2006)). A systematic study of a quencher-based kit, as well as a kit employing the calcium-sensitive FluoForte™ dye, in the presence or absence of Trypan Blue dye, demonstrated exogenous addition of a quencher dye can adversely affect receptor-ligand interaction kinetics. Enhanced assay performance can be achieved by employing the FluoForte™ dye in combination with a cell loading buffer that enhances cell retention and prevents dye extrusion through plasma membrane anion transporters. Incorporating the EL406 Combination Washer Dispenser in the workfl ow provides fast, gentle removal of the serum-containing medium, without loss of cells. Omission of the quencher dye in the assay reduces the potential for non-specific pharmacological interferences in the workfl ow.