Abstract

The precise and simultaneous monitoring of intracellular second messengers, such as cyclic adenosine monophosphate (cAMP) and calcium ions (Ca2+), is fundamental to understanding complex cellular signaling networks. These two messengers often interact in a highly localized and temporally coordinated manner to regulate diverse physiological processes, including gene expression, metabolism, and neuronal activity. To facilitate the study of this critical crosstalk, we developed a stable, monoclonal cell line engineered for the co-expression of genetically encoded fluorescent sensors for both cAMP and Ca2+. The methodology involved stable transfection and rigorous single-cell cloning to ensure uniform, high-level expression of both sensors across the entire population, minimizing experimental variability. Our characterization confirmed that the co-expressed sensors maintain their independent functionality and sensitivity, allowing for real-time, ratiometric imaging of both signaling pathways within the same living cell. This novel tool provides a robust and reliable platform for high-throughput screening and detailed mechanistic studies of signal integration, offering significant advantages over transient co-transfection or sequential measurements. The availability of this stable cell line will be invaluable for dissecting the spatio-temporal dynamics of cAMP and Ca2+ signaling in health and disease.