A high-throughput, multiplex cell death assay using an RNAi screening approach.
A high-throughput, multiplex cell death assay using an RNAi screening approach.
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DOI：
10.1101/pdb.prot080267
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发表时间：
2014-06-02
影响因子：
--
通讯作者：
Simpson, Kaylene J
中科院分区：
文献类型：
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作者：
Falkenberg, Katrina J;Saunders, Darren N;Simpson, Kaylene J
本方案概述了一种高通量、多重细胞死亡试验及其与基因组尺度siRNA筛选相结合的用途，以鉴定与药物合作诱导细胞凋亡的基因。这项名为ApoLive-Glo （Promega）的试验，通过荧光细胞滴度-荧光试剂（包括被活细胞蛋白酶裂解的底物）测量药物处理过的、反向转染的细胞的活力。ApoLive-Glo还使用发光caspase - glo 3/7 caspase激活试验，通过裂解caspase 3和7的数量来定量细胞死亡。多重试验的优势在于，它区分了快速细胞死亡和较慢的半胱天酶活性激活，允许在单个时间点测量同一样品中细胞死亡的不同阶段。同时，高含量成像方案涉及4'，6-二氨基-2-苯基吲哚染色的细胞核被用作一种经济有效的方法来量化载体处理的对照细胞的活力。自动化和机器人液体处理内置于协议中，以提高工作流程的速度并提高再现性。使用这些检测方法的筛选将确定对生存能力至关重要的基因靶标，而不考虑药物治疗，以及在药物存在下导致细胞死亡协同增强的基因靶标。候选靶点的活性可以通过传统的流式细胞术进行验证。
This protocol outlines a high-throughput, multiplex cell death assay and its use in conjunction with a genome-scale siRNA screen to identify genes that cooperate with a drug to induce apoptosis. The assay, ApoLive-Glo (Promega), measures viability of drug-treated, reverse-transfected cells via the fluorescent CellTiter-Fluor reagent, which includes a substrate that is cleaved by a live cell protease. ApoLive-Glo also quantitates cell death by the amount of cleaved caspases 3 and 7 using a luminescent Caspase-Glo 3/7 caspase activation assay. The advantage of the multiplex assay is that it distinguishes rapid cell death from the slower activation of caspase activity, permitting measurement of different stages of cell death in the same sample at a single time point. In parallel, a high-content imaging protocol involving 4',6-diamidino-2-phenylindole-stained nuclei is used as a cost-effective way to quantitate viability of vehicle-treated control cells. Automation and robotic liquid handling are built into the protocol to increase speed of workflow and improve reproducibility. A screen using these assays will identify gene targets that are essential for viability irrespective of drug treatment and gene targets that cause a synergistic enhancement of cell death in the presence of drug. Candidate target activity can then be validated by conventional flow cytometry-based assays.