Affordable luciferase reporter assay for cell-based high-throughput screening

Area of Science:

• Biochemistry
• Molecular Biology
• Cell Biology

Background:

• Firefly luciferase reporter gene assays are crucial for cell-based screening.
• Commercially available reagents for high-throughput screening (HTS) are often prohibitively expensive for academic research.
• There is a need for cost-effective luciferase assay solutions suitable for HTS.

Purpose of the Study:

• To develop an affordable, homemade luciferase assay reagent for cell-based HTS.
• To optimize reagent composition for luminescence intensity and signal stability.
• To validate the reagent's performance in a high-throughput setting.

Main Methods:

• Homemade reagent preparation using individual chemical components.
• Optimization of dithiothreitol (DTT) and D-luciferin concentrations to reduce cost.
• Assessment of signal stability by omitting coenzyme A and adjusting DTT levels.
• Validation using cancer cell lines with a p53-dependent reporter and siRNA knockdown.

Main Results:

• Developed a reagent costing less than one cent per sample.
• Achieved stable signals over a 2-hour measurement period by modifying reagent composition.
• Demonstrated assay quality with Z' factors consistently above 0.5 in validation experiments.
• Successfully monitored p53 transcriptional activity modulation in cancer cells.

Conclusions:

• The developed homemade luciferase assay reagent provides an affordable alternative for cell-based HTS.
• The optimized reagent offers reliable and stable reporter gene readout.
• This cost-effective solution enhances accessibility of HTS for academic laboratories.