title: Multiparameter measurement of caspase 3 activation and apoptotic cell death in NT2 neuronal precursor cells using high-content analysis.
abstract: Caspase activation is a component of a number of neurodegenerative disorders, including stroke. In this study, the authors describe a multiplexed assay for caspase 3 activation, nuclear condensation, and cell viability in a neuronal precursor cell line Ntera-2, injured with staurosporine and etoposide. Using a high-content screening approach, cells were identified by staining with the nuclear stain Hoechst 33342; cell viability was measured by staining cells with YoPro-1, which is taken up by damaged cells but excluded from healthy cells; and caspase 3/7 activation was detected using the cell-permeable probe PhiPhi-Lux, which becomes fluorescent when cleaved by active caspase 3 or 7. These 3 dyes were detected simultaneously using a 4-band pass filter set on a Cellomics Array scan. The authors used peptide-fmk inhibitors selective for a variety of caspases, demonstrating that the injury is mediated primarily through caspase 3 or 7, although other caspases or related proteases may play a minor role. The general caspase inhibitor zVAD-fmkwas able to block cell death and caspase activation with the highest potency. The caspase 3 selective inhibitor DEVD-fmkwas almost as potent as zVAD-fmk; other peptide caspase inhibitors displayed only modest inhibition of cell death. This assay was also used as a high-content screening tool for the evaluation of novel caspase 3 inhibitors for the potential treatment of degenerative disorders.
authors.affiliation: Wyeth Research CN8000 Princeton, NJ 08543, USA. Fennelm@wyeth.com
authors.forename: Myles
authors.initials: M
authors.lastname: Fennell
authors.forename: Helen
authors.initials: H
authors.lastname: Chan
authors.forename: Andrew
authors.initials: A
authors.lastname: Wood
mesh_terms: D017209:Apoptosis; D053148:Caspase 3; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism*; D002460:Cell Line; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004789:Enzyme Activation; D009474:Neurons / Q000187:drug effects / Q000201:enzymology* / Q000473:pathology
chemical_list: D061945:Caspase Inhibitors; D015853:Cysteine Proteinase Inhibitors; D053148:Caspase 3; D020169:Caspases