Microplate orbital mixing improves high-throughput cell-based reporter assay readouts

Area of Science:

• Biochemistry
• Cell Biology
• Assay Development

Background:

• Reporter assays are vital for high-throughput screening in drug discovery and biological research.
• Luciferase reporter gene assays offer robust signals, ease of miniaturization, and simple detection.
• Current assay formulations aim to minimize variability but can be limited by factors like incomplete cell lysis.

Purpose of the Study:

• To investigate the impact of incorporating an orbital mixing step into microplate-based luciferase reporter assays.
• To determine if orbital mixing can improve signal generation and reduce variability in cell-based screening.
• To assess the utility of orbital mixing for assays with high cell densities.

Main Methods:

• Utilized commercially available luciferase assay formulations in 96- and 384-well microplate formats.
• Incorporated an orbital mixing step using commercially available mixers before reporter activity measurement.
• Compared reporter readouts and variability between assays with and without orbital mixing.

Main Results:

• Orbital mixing significantly improved reporter readouts in luciferase assays.
• Maximal luciferase signal generation was achieved from high cell density samples with orbital mixing.
• Orbital mixing minimized variability associated with partial cell lysis, thereby enhancing assay precision.

Conclusions:

• Incorporating orbital mixing is a simple yet effective method to enhance signal and precision in cell-based luciferase reporter assays.
• This technique is particularly beneficial for high cell density samples, improving overall assay performance.
• Further optimization with advanced mixers may extend these benefits to smaller well formats like 1536-well plates.