Division of Bioengineering, School of Chemical and Biomedical Engineering, Nanyang Technological University, Singapore.
Biochem Biophys Res Commun. 2012 Feb 24;418(4):641-6. doi: 10.1016/j.bbrc.2012.01.066. Epub 2012 Jan 21.
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.
在这项研究中，我们开发了一种使用荧光共振能量转移（FRET）的生物传感器的高通量内皮细胞凋亡检测方法。在接触凋亡诱导剂紫外线照射或抗癌药物如紫杉醇后，细胞的荧光从绿色变为蓝色。我们通过测量黄色荧光蛋白（YFP）与青色荧光蛋白（CFP）的发射比，将该方法开发为 96 孔板中的高通量测定法，以监测凋亡过程中关键蛋白酶 caspase-3 的激活。该测定的 Z'因子大于 0.5，表明该测定适用于高通量分析。最后，我们应用这种功能性高通量测定法从我们的内部化合物文库中筛选出血管破坏剂（VDA），这些血管破坏剂可诱导内皮细胞凋亡，发现薯蓣皂苷元是一种有效化合物。由于该测定法可以实时和灵敏地检测细胞凋亡，因此它将成为监测活细胞中内皮细胞凋亡和通过高通量筛选识别新的 VDA 候选物的有用工具。
