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Related Experiment Videos

DEVDase detection in intact apoptotic cells using the cell permeant fluorogenic substrate, (z-DEVD)2-cresyl violet.

Brian W Lee1, Gary L Johnson, Sally A Hed

  • 1Immunochemistry Technologies, LLC, Bloomington, MN, USA. brian@immunochemistry.com

Biotechniques
|November 25, 2003
PubMed
Summary

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Researchers developed a new fluorogenic substrate, (z-DEVD)2-cresyl violet, for detecting active caspase-3 and caspase-7 in apoptotic cells. This method allows rapid, site-specific detection of caspase up-regulation in intact cells without washing.

Area of Science:

  • Biochemistry
  • Cell Biology
  • Molecular Biology

Background:

  • Apoptosis, or programmed cell death, is a critical biological process.
  • Caspases are key enzymes that execute apoptosis, and their up-regulation indicates apoptotic activity.
  • Accurate and timely detection of caspase activity is crucial for studying apoptosis.

Purpose of the Study:

  • To develop a novel, cell-permeant fluorogenic substrate for detecting active caspase-3 and caspase-7.
  • To enable rapid, site-specific detection of caspase up-regulation in intact apoptotic cells.
  • To validate the substrate's efficacy in various cell lines and detection methods.

Main Methods:

  • Synthesis of a bisubstituted caspase-3 target sequence, (z-DEVD)2, conjugated to the fluorophore cresyl violet.

Related Experiment Videos

  • Application of the (z-DEVD)2-cresyl violet substrate to induced and noninduced cell cultures.
  • Detection of substrate conversion and caspase activity using fluorescence microscopy and 96-well fluorescence plate reader analysis.

    • Main Results:

    • The (z-DEVD)2-cresyl violet substrate is cell-permeant and allows for direct detection of caspase activity in intact cells.
    • No wash step is required, enabling rapid assessment of caspase up-regulation.
    • A greater than 6-fold increase in fluorescence was observed in induced versus noninduced Jurkat cells, demonstrating significant DEVDase activity.
    • Successful detection of apoptosis induction was achieved in Jurkat, THP-1, and MCF-7 cell lines.

    Conclusions:

    • The (z-DEVD)2-cresyl violet substrate is an effective tool for the rapid, site-specific detection of active caspase-3 and caspase-7 in living cells.
    • This fluorogenic substrate simplifies the process of apoptosis detection, offering a valuable method for biological research.
    • The developed method provides a sensitive and efficient means to study apoptosis induction in various cellular contexts.