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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
PubMed
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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.

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.