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

er dye concentrations), ratiometric dyes offer several advantages over conventional single-wavelength indicators:

Benefits of using the 340/380 nm excitation ratios for Fura-2:
Signal is not dependent on:
-Dye concentration
-Illumination intensity
-Optical path length

The effects of uneven dye loading, dye leakage and photo bleaching are minimized
-Detection can usually be made over a period of 1 hour without significant loss of fluorescence

A more stable signal and better signal-to-noise ratio may be obtained
- Increased sensitivity

Accurate measurements of the intracellular Ca2+ concentration is possible

An alternative approach is a series of no-wash reagents available from Molecular Devices, such as the FlexStation Calcium Assay Kit. These, too, confer a number of potential advantages over conventional Ca2+ indicators.

Problems in a conventional assay protocol that may be reduced with no-wash reagents:
Cells removed from the plates during the washing procedure
Reduced responsiveness (competence) of the cells following washing
Variation in the residual volume of wash buffer, leading to variation in the concentration of test compound
The drop in fluorescence (resulting from the dilution of residual extracellular dye) upon addition of the test compound

In this application note we demonstrate the use of both single-wavelength and dual-wavelength Ca2+ indicators in the FlexStation benchtop scanning fluorometer and the possible benefits of a homogeneous assay system.
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