Live-cell imaging of caspase activation for high-content screening.
Live-cell imaging of caspase activation for high-content screening.
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DOI：
10.1177/1087057109343207
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发表时间：
2009-09
影响因子：
--
通讯作者：
Djaballah H
中科院分区：
文献类型：
--
作者：
Antczak C;Takagi T;Ramirez CN;Radu C;Djaballah H
半胱天冬酶是执行程序性细胞死亡的核心，其激活构成了细胞凋亡的生化标志。在这篇文章中，我们报告了利用DEVD-NucView 488 ™荧光底物的高含量测定方法的成功适应，并且首次显示了药物或siRNA诱导的活细胞中的半胱天冬酶激活。发现荧光底物在几天的暴露期内是无毒的;在此期间，我们展示了同一细胞群的半胱天冬酶激活随时间的函数的自动成像和定量。单独或与抑制剂Z-VAD-FMK组合的抗凋亡蛋白Bcl-XL的过表达减弱了暴露于阿霉素、依托泊苷或细胞死亡siRNA的HeLa细胞中的半胱天冬酶活化。我们的方法针对两种充分表征的NSCLC细胞系进一步验证，所述NSCLC细胞系报告为对厄洛替尼敏感（H3255）或难治性（H2030）;其中我们显示观察到H3255的差异时间依赖性活化，并且H2030没有显著变化，与它们各自的化学敏感性特征一致。总之，我们的结果证明了使用这种新适应和验证的高含量测定来筛选化学或RNAi文库以鉴定先前未发现的活细胞中凋亡机制的增强子和抑制子的可行性。
Caspases are central to the execution of programmed cell death and their activation constitutes the biochemical hallmark of apoptosis. In this article, we report the successful adaptation of a high content assay method utilizing the DEVD-NucView488™ fluorogenic substrate, and for the first time, we show caspase activation in live cells induced either by drugs or siRNA. The fluorogenic substrate was found to be non-toxic over an exposure period of several days; during which we demonstrate automated imaging and quantification of caspase activation of the same cell population as a function of time. Overexpression of the anti-apoptotic 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. Our method was further validated against two well characterized NSCLC cell lines reported to be sensitive (H3255) or refractory (H2030) to Erlotinib; where we show a differential time dependent activation was observed for H3255 and no significant changes in H2030, consistent with their respective chemosensitivity profile. In summary, our 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.
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影响因子：
4.8
作者：
Cen, Hui;Mao, Fei;Firestone, Gary L.
通讯作者：
Firestone, Gary L.
DOI：
10.1016/j.jasms.2007.08.007
发表时间：
2007-11-01
影响因子：
3.2
作者：
Gabelica, Valerie;Rosu, Frederic;Dugourd, Philippe
通讯作者：
Dugourd, Philippe
DOI：
10.1016/j.bbamcr.2008.01.009
发表时间：
2008-05-01
影响因子：
5.1
作者：
Jones, Sarah;Martel, Cecile;Howl, John
通讯作者：
Howl, John
影响因子：
4.8
作者：
Thornberry, NA;Ranon, TA;Nicholson, DW
通讯作者：
Nicholson, DW
影响因子：
--
作者：
Naqvi, T;Lim, A;Eglen, RM
通讯作者：
Eglen, RM