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Monitoring Receptor-Mediated Changes in [Ca2+]i Using Single-Wavelength and Dual-Wavelength Indicators on the FlexStation Workstation


BACKGROUND
CHO-M1 cells contain endogenous nucleotide receptors (P2Y2)3,4 that are linked to activation of phospholipase C, generation of inositol phosphates (IPs), activation of protein kinase C (PKC) and release of intracellular Ca2+ stores5. Purinoceptors are expressed in the vast majority of cells and tissues, where they control a wide range of physiological processes. To date, five subtypes of the P2Y receptor6 have been identified and clonedP2Y1, P2Y2, P2Y4, P2Y6 and P2Y11. The principal physiological ligands for the P2Y2-receptor are UTP and ATP; they show similar potencies7. One important functional role of the P2Y2-receptor is the control of chloride ion fluxes in airway epithelia and mucociliary clearance in the lung, and thus may play a role in cystic fibrosis and other airway disorders.

Novel ligands targeting P2Y-receptors represent potential therapeutic agents and the ability to screen such compounds reliably and reproducibly has been demonstrated using FlexStation8.

Using Z-factor analysis9 and comparison of EC50 estimates and signal magnitude, we show the benefits of ratiometric Ca2+ dyes and homogeneous assay formats over conventional Ca2+ indicators.


METHODS
Cell Preparation
CHO cells (passage number > 40) were routinely grown in 75 cm2 flasks as a monolayer in Hams-F12 medium supplemented with 10% foetal bovine serum and 5 g/mL G418, and maintained at 37˚C in a CO2 incubator. Cells were passaged 1:10 every 2-3 days.

Measurement of [Ca2+]i
CHO cells were seeded into black-walled, clear-base 96-well plates (Costar UK) at
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