LucFlow: A method to measure Luciferase reporter expression in single cells.

Area of Science:

• Molecular Biology
• Genetics
• Cell Biology

Background:

• Reporter assays are crucial for studying gene regulation by measuring reporter protein expression driven by regulatory elements.
• Existing methods range from low-throughput single-cell techniques to high-throughput bulk assays like Luciferase assays.

Purpose of the Study:

• To develop a high-throughput, single-cell method for accurate reporter gene expression measurement.
• To improve upon the limitations of traditional bulk Luciferase assays.

Main Methods:

• Developed Luciferase Reporter Assay using Flow-Cytometry (LucFlow) to measure single-cell reporter expression via immunostaining.
• Optimized and validated LucFlow using a murine cell line with integrated reporter constructs.
• Incorporated internal controls by spiking samples with stained, distinguishable cells to normalize for experimental variations.

Main Results:

• LucFlow provides single-cell measurements comparable to bulk assays but with higher accuracy.
• Identified that dead cells lack detectable Luciferase, leading to underestimation in bulk assays.
• Demonstrated LucFlow's ability to exclude dead cells, enhancing accuracy and precision in reporter expression quantification.

Conclusions:

• LucFlow enables accurate and precise high-throughput measurement of reporter gene expression at the single-cell level.
• The method addresses the underestimation issue in bulk assays by excluding non-viable cells.
• LucFlow offers a significant advancement for gene regulation research by providing more reliable data.