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

MPS-Authors
http://pubman.mpdl.mpg.de/cone/persons/resource/persons81802

Bartfeld,  Sina
Department of Molecular Biology, Max Planck Institute for Infection Biology, Max Planck Society;

http://pubman.mpdl.mpg.de/cone/persons/resource/persons81935

Hess,  Simone
Department of Molecular Biology, Max Planck Institute for Infection Biology, Max Planck Society;

http://pubman.mpdl.mpg.de/cone/persons/resource/persons81804

Bauer,  Bianca
Department of Molecular Biology, Max Planck Institute for Infection Biology, Max Planck Society;

http://pubman.mpdl.mpg.de/cone/persons/resource/persons82028

Machuy,  Nikolaus
Department of Molecular Biology, Max Planck Institute for Infection Biology, Max Planck Society;

http://pubman.mpdl.mpg.de/cone/persons/resource/persons82078

Ogilvie,  Lesley A.
Department of Molecular Biology, Max Planck Institute for Infection Biology, Max Planck Society;

http://pubman.mpdl.mpg.de/cone/persons/resource/persons82047

Meyer,  Thomas F.
Department of Molecular Biology, Max Planck Institute for Infection Biology, Max Planck Society;

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Fulltext (public)

BMC_Cell_Biol_2010_11_21.pdf
(Publisher version), 2MB

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Citation

Bartfeld, S., Hess, S., Bauer, B., Machuy, N., Ogilvie, L. A., Schuchhardt, J., et al. (2010). High-throughput and single-cell imaging of NF-kappa B oscillations using monoclonal cell lines. BMC Cell Biology, 11: 21.


Cite as: http://hdl.handle.net/11858/00-001M-0000-000E-C00E-C
Abstract
Background: The nuclear factor-kappa B (NF-kappa B) 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-kappa B system has been associated with a number of disease states, including cancer. Therefore, insight into the regulation of NF-kappa B activation has crucial medical relevance, holding promise for novel drug target discovery. Transcription of NF-kappa B-induced genes is regulated by differential dynamics of single NF-kappa B subunits, but only a few methods are currently being applied to study dynamics. In particular, while oscillations of NF-kappa B activation have been observed in response to the cytokine tumor necrosis factor alpha (TNF alpha), little is known about the occurrence of oscillations in response to bacterial infections. Results: To quantitatively assess NF-kappa B dynamics we generated human and murine monoclonal cell lines that stably express the NF-kappa B 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-kappa B 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-kappa B kinetics in single cells and cell populations. Using a single system, novel factors modulating NF-kappa B 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.