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| Content Provider | World Health Organization (WHO)-Global Index Medicus |
|---|---|
| Author | Falkenberg, Katrina J. Saunders, Darren N. Simpson, Kaylene J. |
| Description | Country affiliation: Australia Author Affiliation: Falkenberg KJ ( Victorian Centre for Functional Genomics, Peter MacCallum Cancer Centre, East Melbourne, Victoria 3002, Australia Department of Pathology, The University of Melbourne, Parkville, Victoria 3052, Australia.); Saunders DN ( Cancer Research Program, The Kinghorn Cancer Centre, Garvan Institute of Medical Research, Darlinghurst, New South Wales 2010, Australia St. Vincent's Clinical School, University of New South Wales Medicine, Sydney, New South Wales 2000, Australia.); Simpson KJ ( Victorian Centre for Functional Genomics, Peter MacCallum Cancer Centre, East Melbourne, Victoria 3002, Australia Department of Pathology, The University of Melbourne, Parkville, Victoria 3052, Australia Sir Peter MacCallum Department of Oncology, The University of Melbourne, Parkville, Victoria 3052, Australia.) |
| Abstract | 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. |
| File Format | HTM / HTML |
| ISSN | 19403402 |
| Issue Number | 6 |
| Volume Number | 2014 |
| e-ISSN | 15596095 |
| Journal | Cold Spring Harbor Protocols |
| Language | English |
| Publisher | Cold Spring Harbor Laboratory Press |
| Publisher Date | 2014-06-02 |
| Publisher Place | United States |
| Access Restriction | One Nation One Subscription (ONOS) |
| Subject Keyword | Discipline Clinical Laboratory Techniques Cell Death Cell Survival Drug Effects Cytological Techniques Methods Drug Evaluation, Preclinical High-throughput Screening Assays Rna Interference Cell Line Fluorescent Dyes Analysis Humans Staining And Labeling Journal Article Research Support, Non-u.s. Gov't |
| Content Type | Text |
| Resource Type | Article |
| Subject | Biochemistry, Genetics and Molecular Biology |
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