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J Biomol Screen. 2009 Sep;14(8):956-69. doi: 10.1177/1087057109343207. Epub 2009 Sep 2.

Live-cell imaging of caspase activation for high-content screening.

Author information

1
High Throughput Screening Core Facility, Memorial Sloan-Kettering Cancer Center, New York, NY 10065, USA.

Abstract

Caspases are central to the execution of programmed cell death, and their activation constitutes the biochemical hallmark of apoptosis. In this article, the authors report the successful adaptation of a high-content assay method using the DEVDNucView488 fluorogenic substrate, and for the first time, they show caspase activation in live cells induced by either drugs or siRNA. The fluorogenic substrate was found to be nontoxic over an exposure period of several days, during which the authors demonstrate automated imaging and quantification of caspase activation of the same cell population as a function of time. Overexpression of the antiapoptotic protein Bcl-XL, alone or in combination with the inhibitor Z-VAD-FMK, attenuated caspase activation in HeLa cells exposed to doxorubicin, etoposide, or cell death siRNA. This method was further validated against 2 well-characterized NSCLC cell lines reported to be sensitive (H3255) or refractory (H2030) to erlotinib, where the authors show a differential time-dependent activation was observed for H3255 and no significant changes in H2030, consistent with their respective chemosensitivity profile. In summary, the results demonstrate the feasibility of using this newly adapted and validated high-content assay to screen chemical or RNAi libraries for the identification of previously uncovered enhancers and suppressors of the apoptotic machinery in live cells.

PMID:
19726787
PMCID:
PMC3613133
DOI:
10.1177/1087057109343207
[Indexed for MEDLINE]
Free PMC Article

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