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Ratiometric Ca²+ measurements using the FlexStation® Scanning Fluorometer.

Ian C B Marshall¹, Izzy Boyfield, Shaun McNulty

¹Neurology Centre of Excellence for Drug Discovery, GlaxoSmithKline Pharmaceuticals Ltd., Harlow, Essex, UK.

Methods in Molecular Biology (Clifton, N.J.)

|February 23, 2011

Summary

The Fluorometric Imaging Plate Reader (FLIPR) is widely used for high-throughput intracellular Ca(2+) measurements. While visible light probes are common, ratiometric dyes offer advantages for specific research needs like improved signal-to-noise ratio.

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Area of Science:

Biochemistry
Cell Biology
Pharmacology

Background:

High-throughput screening relies on accurate intracellular Ca(2+) measurements.
The Fluorometric Imaging Plate Reader (FLIPR) is a standard tool for these assays.
FLIPR typically uses single-wavelength fluorescent probes with visible light excitation.

Purpose of the Study:

To discuss the advantages and limitations of FLIPR for high-throughput Ca(2+) measurements.
To explore the utility of ratiometric dyes in specific research scenarios.
To provide context for selecting appropriate fluorescent probes for cellular assays.

Main Methods:

Review of FLIPR technology and its applications in commercial settings.
Comparison of single-wavelength visible light probes versus ratiometric probes.

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Discussion of factors influencing probe selection, including dye leakage, photobleaching, and signal-to-noise ratio.

Main Results:

FLIPR's rapid kinetics, reliability, and automation compatibility make it suitable for many industrial applications.
Visible light probes offer benefits like reduced autofluorescence and higher absorbance.
Ratiometric dyes may be preferred when dye leakage, photobleaching, or signal-to-noise ratio are critical concerns.

Conclusions:

FLIPR is a versatile platform for high-throughput Ca(2+) assays.
The choice between visible light and ratiometric probes depends on specific experimental requirements.
Understanding probe characteristics is crucial for optimizing cellular assay performance.