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Cell-Based Assays to Identify Modulators of Nrf2/ARE Pathway
Zhengxi Wei1, Jinghua Zhao1, Li Zhang1
1National Center for Advancing Translational Sciences, National Institutes of Health, Bethesda, MD, USA.
Methods in Molecular Biology (Clifton, N.J.)
|March 16, 2022
Summary
This study details two cell-based assays for detecting oxidative stress via the Nrf2/ARE pathway. These high-throughput screening methods efficiently identify chemicals that modulate cellular oxidative stress signaling.
Area of Science:
- Biochemistry
- Cell Biology
- Toxicology
Background:
- Cellular oxidative stress is a critical factor in various diseases.
- The nuclear factor erythroid 2-related factor (Nrf2) and antioxidant response element (ARE) pathway is a key regulator of the cellular antioxidant response.
- Identifying modulators of this pathway is crucial for understanding and treating oxidative stress-related conditions.
Purpose of the Study:
- To describe two novel cell-based assays for quantifying Nrf2/ARE pathway activation.
- To establish these assays in a high-throughput screening (HTS) format for large-scale chemical library screening.
- To provide a detailed protocol for assay implementation, including cell handling, preparation, and instrumentation.
Main Methods:
- Development and validation of two distinct cell-based assays measuring Nrf2/ARE pathway activity.
- Implementation of a quantitative high-throughput screening (HTS) approach.
- Detailed description of cell culture, assay reagent preparation, instrument setup, and data acquisition procedures.
Main Results:
- The described assays provide a quantitative measure of Nrf2/ARE pathway activation.
- The HTS format enables the efficient screening of large chemical collections.
- The assays are robust and suitable for identifying compounds that induce or inhibit oxidative stress signaling.
Conclusions:
- The developed cell-based Nrf2/ARE assays are effective tools for oxidative stress research.
- These HTS assays facilitate the discovery of novel chemicals impacting cellular redox homeostasis.
- The detailed protocols ensure reproducibility and applicability in various research settings.
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