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Updated: May 25, 2026
A Simple Bioassay for the Evaluation of Vascular Endothelial Growth Factors
Published on: March 15, 2016
A high-throughput fluorescence resonance energy transfer (FRET)-based endothelial cell apoptosis assay and its
Xiaoming Zhu1, Afu Fu, Kathy Qian Luo
1Division of Bioengineering, School of Chemical and Biomedical Engineering, Nanyang Technological University, Singapore.
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.
Insights
We developed a high-throughput assay to detect endothelial cell apoptosis using a FRET biosensor. This method identified dioscin as a potential vascular disrupting agent (VDA) for cancer therapy.
Area of Science:
- Biochemistry
- Cell Biology
- Pharmacology
Background:
- Endothelial cell apoptosis is crucial for regulating vascular integrity and is a target for cancer therapies.
- Existing methods for apoptosis detection often lack the throughput required for large-scale screening.
Purpose of the Study:
- To develop a high-throughput assay for monitoring endothelial cell apoptosis.
- To screen for novel vascular disrupting agents (VDAs) using this assay.
Main Methods:
- A fluorescence resonance energy transfer (FRET)-based biosensor was engineered to detect caspase-3 activation, a key apoptosis marker.
- The assay was optimized for 96-well plates, measuring the emission ratio of yellow fluorescent protein (YFP) to cyan fluorescent protein (CFP).
- The assay's suitability for high-throughput screening was validated with a Z' factor above 0.5.
Main Results:
- The FRET biosensor successfully detected apoptosis induced by UV irradiation and anticancer drugs (e.g., paclitaxel), showing a green-to-blue fluorescence shift.
- High-throughput screening of an in-house compound library identified dioscin as a hit compound that induces endothelial cell apoptosis.
- The assay demonstrated real-time and sensitive detection of apoptosis in living cells.
Conclusions:
- A robust, high-throughput FRET-based assay for endothelial cell apoptosis has been established.
- This assay is effective for identifying potential VDAs and can be applied to drug discovery for cancer therapy.
- The developed assay serves as a valuable tool for real-time monitoring of endothelial cell apoptosis.
