Cell-based multiwell assays for the detection of substrate accumulation and oxidation

Area of Science:

• Biochemistry
• Cell Biology
• Metabolic Research

Background:

• Understanding cellular fuel handling is crucial for metabolic research.
• Existing methods for tracking substrate metabolism in cells can be complex or time-consuming.
• Need for robust assays to monitor substrate uptake, storage, and breakdown.

Purpose of the Study:

• To develop and validate novel multiwell assays for real-time detection of substrate accumulation and oxidation in cultured cells.
• To provide adaptable and efficient tools for studying cellular fuel metabolism.
• To facilitate compound screening for metabolic modulators.

Main Methods:

• Real-time monitoring of radiolabeled substrate accumulation using scintillation proximity assays.
• A novel gas-tight multiwell assay for trapping and quantifying (14)CO(2) produced from substrate oxidation.
• Application of assays in cultured human myotubes, adipocytes, and hepatocytes.

Main Results:

• Successful demonstration of both substrate accumulation and oxidation in various human cell types.
• The developed assays are sensitive and suitable for real-time measurements.
• The substrate oxidation assay effectively captures and quantifies (14)CO(2) in a gas-tight system.

Conclusions:

• The described multiwell assays provide efficient, time-saving, and adaptable methods for in vitro studies of cellular fuel handling.
• These assays are valuable tools for basic research and high-throughput compound screening in metabolic studies.
• The novel oxidation assay enhances the ability to study metabolic pathways in intact cells.