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Measuring calcium signaling using genetically targetable fluorescent indicators.

Amy E Palmer1, Roger Y Tsien

  • 1Department of Chemistry and Biochemistry, University of Colorado, Boulder, Colorado 80309-0215, USA. amy.palmer@colorado.edu

Nature Protocols
|April 5, 2007
PubMed
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This protocol details using genetically encoded calcium indicators (GECIs) for quantitative Ca2+ dynamics measurement. It focuses on cameleon indicators and provides methods for imaging, data processing, and calibration, especially for organellar Ca2+ in mammalian cells.

Area of Science:

  • Biochemistry
  • Cell Biology
  • Biophysics

Background:

  • Genetically encoded Ca2+ indicators (GECIs) enable quantitative measurement of intracellular Ca2+ dynamics.
  • Various GECIs exist, each suited for specific experimental conditions, including organellar localization in mammalian cells.

Purpose of the Study:

  • To provide a protocol for using GECIs, specifically cameleon (a FRET-based indicator), for Ca2+ imaging.
  • To detail experimental setup, data processing, and calibration strategies for GECIs.
  • To address challenges in measuring organellar Ca2+ and offer solutions.

Main Methods:

  • Utilizes cameleon, a genetically encoded FRET-based Ca2+ indicator.
  • Describes Ca2+ imaging experiment setup and execution.
  • Outlines offline data processing, including background correction and FRET ratio conversion to Ca2+ concentrations.

Related Experiment Videos

  • Explains calibration strategies for organellar Ca2+ measurements.

    • Main Results:

    • The protocol enables quantitative measurement of Ca2+ dynamics using GECIs.
    • It provides a framework for setting up Ca2+ imaging experiments and processing data.
    • Challenges and calibration methods for organellar Ca2+ are highlighted.

    Conclusions:

    • This protocol facilitates the effective use of GECIs for studying Ca2+ dynamics in various cellular compartments.
    • It offers practical guidance for researchers, particularly those investigating organellar Ca2+ in mammalian cells.
    • The methods described aid in accurate quantitative analysis of Ca2+ signaling.