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Detection of programmed cell death in cells exposed to genotoxic agents using a caspase activation assay

Madhu Gupta1, Madhumita Santra, Patrick P Koty

  • 1Department of Pediatrics, Wake Forest University School of Medicine, Winston-Salem, NC, USA.

Methods in Molecular Biology (Clifton, N.J.)
|March 14, 2014
PubMed

Insights

Toxins can cause DNA damage, leading cells to programmed cell death (PCD). This study details detecting activated caspases, key enzymes in PCD, within intact cells using fluorescent probes and microscopy.

Area of Science:

  • Cellular biology
  • Molecular toxicology
  • Biochemistry

Background:

  • Environmental toxins and endogenous agents frequently induce DNA damage in cells.
  • Cells possess repair mechanisms, but severe damage triggers programmed cell death (PCD).
  • Caspases, a family of cysteine proteases, are critical executioners of PCD, activated late in the pathway.

Purpose of the Study:

  • To establish a method for detecting activated caspases in intact cells.
  • To visualize caspase activation preceding morphological changes in PCD.

Main Methods:

  • Utilizing fluorochrome-conjugated caspase inhibitors for detection.
  • Employing fluorescence microscopy for analysis and documentation.
  • Applying the method to intact cells to observe early caspase activation.

Main Results:

  • Demonstrated successful detection of activated caspases in intact cells.
  • Visualized caspase activation as an early event in the PCD pathway.
  • Provided a protocol for quantitative and qualitative assessment of caspase activity.

Conclusions:

  • The described method allows for early and specific detection of caspase activation during programmed cell death.
  • This technique aids in understanding the role of caspases in cellular responses to DNA damage.
  • Fluorescence-based caspase detection offers a valuable tool for toxicological and cell death research.

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