A high-throughput, multiplex cell death assay using an RNAi screening approach.

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.