Detection of programmed cell death in cells exposed to genotoxic agents using a caspase activation assay.
Many environmental toxins cause DNA damage. Cells that have sustained significant DNA damage must attempt to repair the damage prior to replication, in which aberrant base incorporation can result in an irreversible mutation. If a cell cannot repair the damage, however, it may 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 in the PCD pathway, when a cell can no longer avoid cell death, but earlier than other PCD markers, such as 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 using fluorescent microscopy.
Gehring
Michael E
ME
Department of Pediatrics, Wake Forest University School of Medicine, Winston-Salem, NC, USA.
Koty
Patrick P
PP
D000818:Animals; D017209:Apoptosis; D001681:Biological Assay; D061945:Caspase Inhibitors; D020169:Caspases / Q000032:analysis* / Q000378:metabolism; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004249:DNA Damage; D053938:DNA Fragmentation; D004789:Enzyme Activation; D006801:Humans; D008856:Microscopy, Fluorescence / Q000379:methods*; D009153:Mutagens / Q000633:toxicity
D061945:Caspase Inhibitors; D015853:Cysteine Proteinase Inhibitors; D009153:Mutagens; D020169:Caspases