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

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.

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Genre: Journal Article
Alternative Title : BMC Cell Biol.

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© 2010 Bartfeld et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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 Creators:
Bartfeld, Sina1, Author           
Hess, Simone1, Author           
Bauer, Bianca1, Author           
Machuy, Nikolaus1, Author           
Ogilvie, Lesley A.1, Author           
Schuchhardt, Johannes, Author
Meyer, Thomas F.1, Author           
Affiliations:
1Department of Molecular Biology, Max Planck Institute for Infection Biology, Max Planck Society, ou_1664147              

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 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.

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Language(s): eng - English
 Dates: 2010-03-16
 Publication Status: Published in print
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 Table of Contents: -
 Rev. Type: Peer
 Identifiers: eDoc: 533652
ISI: 000276333600001
 Degree: -

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Title: BMC Cell Biology
  Alternative Title : BMC Cell Biol.
Source Genre: Journal
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Pages: - Volume / Issue: 11 Sequence Number: 21 Start / End Page: - Identifier: ISSN: 1471-2121