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Cell-Based Assay for Identifying the Modulators of Antioxidant Response Element Signaling Pathway
Jinghua Zhao1, Sunita J Shukla2, Menghang Xia3
1National Center for Advancing Translational Sciences, National Institutes of Health, Building C, MSC: 3375, 9800 Medical Center Drive, Bethesda, MD, 20892, USA.
Abstract:
The antioxidant response element (ARE) signaling pathway plays an important role in the amelioration of cellular oxidative stress. Thus, assays that detect this pathway can be useful for identifying chemicals that induce or inhibit oxidative stress signaling. The focus of this chapter is to describe a cell-based ARE assay in a quantitative high-throughput screening (qHTS) format to test a large collection of compounds that induce nuclear factor erythroid 2-related factor (Nrf2)/ARE signaling. The assay is described through cell handling, assay preparation, and instrument usage.
Insights
This study details a cell-based assay to screen compounds affecting the antioxidant response element (ARE) pathway. This method aids in identifying chemicals that modulate oxidative stress signaling via the Nrf2/ARE pathway.
Area of Science:
- Cellular biology
- Molecular signaling
- Drug discovery
Background:
- Cellular oxidative stress is a critical factor in various diseases.
- The antioxidant response element (ARE) signaling pathway is key in mitigating oxidative stress.
- Identifying modulators of this pathway is crucial for therapeutic development.
Purpose of the Study:
- To describe a cell-based assay for detecting the ARE signaling pathway.
- To adapt this assay into a quantitative high-throughput screening (qHTS) format.
- To enable the screening of large compound libraries for Nrf2/ARE pathway modulators.
Main Methods:
- Development of a cell-based assay focused on the ARE signaling pathway.
- Implementation of a quantitative high-throughput screening (qHTS) format.
- Detailed procedures covering cell handling, assay preparation, and instrument operation.
Main Results:
- The described qHTS assay effectively measures Nrf2/ARE pathway activity.
- The assay format is suitable for testing large collections of chemical compounds.
- The methodology provides a quantitative readout for pathway induction or inhibition.
Conclusions:
- The cell-based qHTS ARE assay is a valuable tool for oxidative stress research.
- This assay facilitates the discovery of novel compounds targeting the Nrf2/ARE pathway.
- The described methodology supports efficient screening for chemicals impacting cellular redox balance.
