title: A high-throughput fluorescence resonance energy transfer (FRET)-based endothelial cell apoptosis assay and its application for screening vascular disrupting agents.
abstract: In this study, we developed a high-throughput endothelial cell apoptosis assay using a fluorescence resonance energy transfer (FRET)-based biosensor. After exposure to apoptotic inducer UV-irradiation or anticancer drugs such as paclitaxel, the fluorescence of the cells changed from green to blue. We developed this method into a high-throughput assay in 96-well plates by measuring the emission ratio of yellow fluorescent protein (YFP) to cyan fluorescent protein (CFP) to monitor the activation of a key protease, caspase-3, during apoptosis. The Z' factor for this assay was above 0.5 which indicates that this assay is suitable for a high-throughput analysis. Finally, we applied this functional high-throughput assay for screening vascular disrupting agents (VDA) which could induce endothelial cell apoptosis from our in-house compounds library and dioscin was identified as a hit. As this assay allows real time and sensitive detection of cell apoptosis, it will be a useful tool for monitoring endothelial cell apoptosis in living cell situation and for identifying new VDA candidates via a high-throughput screening.
authors.affiliation: Division of Bioengineering, School of Chemical and Biomedical Engineering, Nanyang Technological University, Singapore.
authors.forename: Xiaoming
authors.initials: X
authors.lastname: Zhu
authors.forename: Afu
authors.initials: A
authors.lastname: Fu
authors.forename: Kathy Qian
authors.initials: KQ
authors.lastname: Luo
mesh_terms: D020533:Angiogenesis Inhibitors / Q000302:isolation & purification* / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects*; D015374:Biosensing Techniques; D053148:Caspase 3 / Q000032:analysis / Q000096:biosynthesis; D002460:Cell Line; D055808:Drug Discovery / Q000379:methods*; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D057166:High-Throughput Screening Assays; D061307:Human Umbilical Vein Endothelial Cells / Q000187:drug effects* / Q000201:enzymology / Q000528:radiation effects; D006801:Humans; D017239:Paclitaxel / Q000494:pharmacology; D014466:Ultraviolet Rays
chemical_list: D020533:Angiogenesis Inhibitors; D053148:Caspase 3; D017239:Paclitaxel