High-throughput and single-cell imaging of NF-κB oscillations using monoclonal cell lines

Abstract Background The nuclear factor-κB (NF-κB) family of transcription factors plays a role in a wide range of cellular processes including the immune response and cellular growth. In addition, deregulation of the NF-κB system has been associated with a number of disease states, including cancer. Therefore, insight into the regulation of NF-κB activation has crucial medical relevance, holding promise for novel drug target discovery. Transcription of NF-κB-induced genes is regulated by differential dynamics of single NF-κB subunits, but only a few methods are currently being applied to study dynamics. In particular, while oscillations of NF-κB activation have been observed in response to the cytokine tumor necrosis factor α (TNFα), little is known about the occurrence of oscillations in response to bacterial infections. Results To quantitatively assess NF-κB dynamics we generated human and murine monoclonal cell lines that stably express the NF-κB subunit p65 fused to GFP. Furthermore, a high-throughput assay based on automated microscopy coupled to image analysis to quantify p65-nuclear translocation was established. Using this assay, we demonstrate a stimulus- and cell line-specific temporal control of p65 translocation, revealing, for the first time, oscillations of p65 translocation in response to bacterial infection. Oscillations were detected at the single-cell level using real-time microscopy as well as at the population level using high-throughput image analysis. In addition, mathematical modeling of NF-κB dynamics during bacterial infections predicted masking of oscillations on the population level in asynchronous activations, which was experimentally confirmed. Conclusions Taken together, this simple and cost effective assay constitutes an integrated approach to infer the dynamics of NF-κB kinetics in single cells and cell populations. Using a single system, novel factors modulating NF-κB can be identified and analyzed, providing new possibilities for a wide range of applications from therapeutic discovery and understanding of disease to host-pathogen interactions.