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Measuring Caspase Activity Using a Fluorometric Assay or Flow Cytometry
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Imaging-based methods for assessing caspase activity in single cells.

Melissa J Parsons1, Markus Rehm2, Lisa Bouchier-Hayes1

  • 1Center for Cell and Gene Therapy, Baylor College of Medicine, Houston, Texas 77030 Department of Pediatrics-Hematology, Baylor College of Medicine, Houston, Texas 77030.

Cold Spring Harbor Protocols
|January 7, 2015
PubMed
Summary
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This study introduces advanced imaging techniques to visualize caspase activation and activity during apoptosis. These methods, including Bimolecular Fluorescence Complementation (BiFC) and Förster Resonance Energy Transfer (FRET), offer detailed insights into cell death pathways.

Area of Science:

  • Cell Biology
  • Molecular Biology
  • Biochemistry

Background:

  • Caspases are key proteases mediating apoptosis, divided into initiator and executioner types.
  • Initiator caspases activate via induced proximity, while executioner caspases activate through cleavage.
  • Understanding caspase activation dynamics is crucial for dissecting cell death signaling.

Purpose of the Study:

  • To introduce and explain BiFC and FRET imaging techniques for studying caspase activation and activity.
  • To highlight the utility of single-cell imaging in dissecting asynchronous apoptosis pathways.
  • To provide insights into the kinetic and positional aspects of cell death signal transduction.

Main Methods:

  • Bimolecular fluorescence complementation (BiFC) for measuring initiator caspase induced proximity.

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  • Förster resonance energy transfer (FRET) for real-time investigation of executioner caspase substrate cleavage.
  • Single-cell imaging coupled with parallel detection of apoptosis hallmarks.

    • Main Results:

    • BiFC and FRET enable visualization of distinct caspase activation mechanisms.
    • These techniques allow for the ordering and dissection of complex caspase pathways.
    • Quantitative kinetic and positional data on cell death signaling can be obtained.

    Conclusions:

    • BiFC and FRET are powerful tools for single-cell analysis of caspase activation and activity.
    • These imaging methods provide critical insights into the regulation of apoptosis.
    • Advanced imaging is essential for a comprehensive understanding of cell death processes.