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High Throughput Screening Assessment of Reactive Oxygen Species (ROS) Generation using Dihydroethidium (DHE) Fluorescence Dye
Published on: January 19, 2024
Reactive oxygen species and cellular oxygen sensing.
Timothy P Cash1, Yi Pan, M Celeste Simon
1Howard Hughes Medical Institute, University of Pennsylvania, Pennsylvania, USA.
Free Radical Biology & Medicine
|September 26, 2007
Summary
Organisms adapt to low oxygen (hypoxia) using transcriptional programs. Reactive oxygen species (ROS) are crucial for stabilizing hypoxia-inducible factors (HIFs) in mammalian cells, aiding this adaptation.
Area of Science:
- Cellular Biology
- Molecular Biology
- Biochemistry
Background:
- Organisms utilize adaptive transcriptional programs to survive low oxygen (hypoxia).
- Hypoxia-inducible factors (HIFs) regulate genes essential for maintaining energy balance during hypoxia in metazoans.
- HIF regulation involves prolyl hydroxylase activity sensitive to oxygen levels, metabolites, and reactive oxygen species (ROS).
Purpose of the Study:
- To investigate the role of ROS in cellular oxygen sensing and HIF stabilization during hypoxia.
- To explore the mechanisms by which ROS influence prolyl hydroxylase activity and HIF stabilization.
Main Methods:
- Utilized fluorescence energy transfer resonance (FRET)-based methods for ROS measurement.
- Employed genetic models with mutations in mitochondrial electron transport chain components.
- Developed an in vivo prolyl hydroxylase reporter.
Main Results:
- Demonstrated that functional mitochondrial electron transport and ROS production are necessary for HIF stabilization in hypoxic mammalian cells.
- Advanced ROS measurement techniques overcome previous challenges associated with ROS instability.
Conclusions:
- ROS play a critical role in mediating HIF stabilization during hypoxia.
- New methodologies and genetic models are facilitating a deeper understanding of ROS-mediated oxygen sensing pathways.
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