Highly sensitive luciferase-based assay with red fluorescent protein expression for accurate quantitative monitoring...

Traditional migration and invasion assays like scratch, Transwell, and Boyden chamber are widely used but have disadvantages such as being time-consuming, lacking real-time monitoring, and relying on endpoint measurements. We addressed these limitations by developing a novel fluorescent and luciferase-based invasion assay.

Three stable cell lines co-expressing the red fluorescent protein dTomato, and secreting luciferase were generated based on Caco-2, MDA-MB-231 and HEK293T cells. Transwell chamber membranes were coated with Matrigel for invasion assay, onto which the modified cells were seeded. To simulate non-invasive and invasive conditions, chambers were incubated for 48 h in FBS-free or FBS-supplemented medium. Following incubation, the Matrigel along with non-invasive cells were removed, and the chambers washed before being transferred into fresh media for 24 h allowing the cells to secrete luciferase. Luciferase activity was measured and compared to traditional cell counting invasion assay, with further confirmations through Z-stacking and microscopic fluorescent imaging.

Our results demonstrated that luciferase activity accurately correlates with cell count. Applying luciferase efficiently quantifies variation in cell invasion with higher sensitivity, hence improving detection of low-level invasion as compared to cell counting techniques based on nuclear staining. The expression of the fluorescent dTomato protein proved ideal for real-time visualization of invading cells.

Overall, using luciferase and dTomato co-expressing cells for invasion assay showed reliable and accurate measurements of variations in cell invasion patterns. Introducing these cells reduced time-consuming steps, improved sensitivity, and endpoints measurements, while being capable of real-time visualization, providing advantages over traditional methods.