Analytical biochemistry · 2025
Highly sensitive luciferase-based assay with red fluorescent protein expression for accurate quantitative monitoring and real-time visualization of cell invasion.
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Christensen Michael Lyngbæk, Kirkegaard Tove, Gögenur Ismail, Diness Frederik, Troelsen Jesper Thorvald, Jessen Stine Bull
Аннотация
BACKGROUND: 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.
MATERIALS AND METHODS: 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.
RESULTS: 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.
CONCLUSION: 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.
MATERIALS AND METHODS: 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.
RESULTS: 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.
CONCLUSION: 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.