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

Simon Lydford & Petra Averbeck, Molecular Devices Limited, U.K.


Introduction
The FlexStation scanning fluorometer integrates sensitive optics, fluid transfer and temperature control, making it ideal for kinetic, cell-based fluorometric assays, such as the measurement of intracellular calcium, membrane potential and intracellular pH.

Calcium (Ca2+) is the most common signal transduction element in cells ranging from bacteria to specialized neurons. Measurement of changes in the concentration of intracellular cations, for example Ca2+, is important in understanding the mechanisms of many cellular processes1. A range of events such as receptor-ligand interactions mediate the elevation of intracellular calcium ([Ca2+]i) levels from ~100nM to ~1mM. This can trigger a number of events, including the release of synaptic transmitters, muscle contraction, hormone secretion, cell movements or apoptosis.

Changes in [Ca2+]i can be measured either with single-wavelength dyes (e.g. Fluo-3 and the FlexStation Calcium Assay Kit) or dual-wavelength ratiometric indicators, such as Fura-2. With single-wavelength dyes the fluorescence intensity is dependent on the Ca2+ concentration whilst ratiometric indicators undergo a spectral shift that is proportional to the Ca2+ concentration. The most common single-wavelength indicators, such as Fluo-3, are excited with visible light and the most common dual-wavelength dyes (Fura-2) require UV light2.

The FlexStation system incorporates a Xenon-lamp light source and dual monochromators that permit the use of essentially all dual-wavelength dyes for functional cellular assays. Although visible-light excitable Ca2+ indicators offer benefits such as reduced interference from sample auto fluorescence and higher absorbance by the dye (permitting the use of low
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