Protocol

Molecular Toxicology Protocols

Volume 1105 of the series Methods in Molecular Biology pp 623-632

Date:

Detection of Programmed Cell Death in Cells Exposed to Genotoxic Agents Using a Caspase Activation Assay

  • Madhu GuptaAffiliated withDepartment of Pediatrics, Wake Forest University School of Medicine
  • , Madhumita SantraAffiliated withDepartment of Pediatrics, Wake Forest University School of Medicine
  • , Patrick P. KotyAffiliated withDepartment of Pediatrics, Wake Forest University School of Medicine Email author 

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Abstract

Many toxins that individuals are exposed to cause DNA damage. Cells that have sustained DNA damage may attempt to repair the damage prior to replication. However, if a cell has sustained serious damage it cannot repair, it will commit suicide through a genetically regulated programmed cell death (PCD) pathway. Crucial to the ultimate execution of PCD is a family of cysteine proteases called caspases. Activation of these enzymes occurs late enough in the PCD pathway that a cell can no longer avoid cell death, but still earlier than PCD-associated morphological changes or DNA fragmentation. This protocol details a method for using fluorochrome-conjugated caspase inhibitors for the detection of activated caspases in intact cells. The analysis and documentation is performed using fluorescence microscopy.

Key words

Apoptosis Programmed cell death (PCD) Caspase Inhibitor Fluorescence DNA damage Genotoxic agent