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High-throughput screening for agonists of ROS production in live human vascular endothelial cells
Tomoya Sasahara1,2, Minako Hoshi1,3
1Department for Brain and Neurodegenerative Disease Research, Institute of Biomedical Research and Innovation, Foundation for Biomedical Research and Innovation at Kobe, CLIK 6F 6-3-7 Minatojima-Minamimachi, Chuo-ku, Kobe 650-0047, Japan.
This study optimized a high-throughput screening protocol to identify agonists that stimulate reactive oxygen species (ROS) production. The method uses a fluorescent probe in endothelial cells to locate ROS generation, aiding future research tools.
Area of Science:
- Biochemistry
- Cell Biology
- Pharmacology
Background:
- Reactive oxygen species (ROS) are crucial physiological molecules with diverse signaling roles.
- Understanding ROS production mechanisms is vital for developing targeted therapeutics and research tools.
- Current methods for identifying ROS-modulating compounds can be limited in throughput and localization specificity.
Purpose of the Study:
- To develop and optimize a high-throughput screening (HTS) protocol for identifying agonists that induce ROS production.
- To establish a method for localizing ROS generation within live human vascular endothelial cells (mitochondria vs. plasma membrane).
- To provide a reliable protocol for advancing ROS-related research and drug discovery.
Main Methods:
- Utilized a fluorescent probe assay for ROS detection in live human vascular endothelial cells.
- Implemented a high-throughput screening approach to efficiently test numerous compounds.
- Developed a protocol to differentiate ROS production sites (mitochondria and plasma membrane).
Main Results:
- Successfully optimized a protocol for HTS of ROS production agonists.
- Demonstrated the capability to distinguish ROS generation in mitochondria versus the plasma membrane.
- Established a robust method for identifying compounds that modulate ROS levels in specific cellular compartments.
Conclusions:
- The presented optimized protocol enables efficient HTS for ROS production agonists.
- This method provides a valuable tool for investigating ROS signaling and identifying novel therapeutic targets.
- The ability to localize ROS production enhances the understanding of cellular responses to oxidative stress.
