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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: Zeitschriftenartikel
Alternativer Titel : BMC Cell Biol.

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BMC_Cell_Biol_2010_11_21.pdf (Verlagsversion), 2MB
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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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Bartfeld, Sina1, Autor           
Hess, Simone1, Autor           
Bauer, Bianca1, Autor           
Machuy, Nikolaus1, Autor           
Ogilvie, Lesley A.1, Autor           
Schuchhardt, Johannes, Autor
Meyer, Thomas F.1, Autor           
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1Department of Molecular Biology, Max Planck Institute for Infection Biology, Max Planck Society, ou_1664147              

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 Zusammenfassung: 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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Sprache(n): eng - English
 Datum: 2010-03-16
 Publikationsstatus: Erschienen
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 Art der Begutachtung: Expertenbegutachtung
 Identifikatoren: eDoc: 533652
ISI: 000276333600001
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Titel: BMC Cell Biology
  Alternativer Titel : BMC Cell Biol.
Genre der Quelle: Zeitschrift
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Ort, Verlag, Ausgabe: -
Seiten: - Band / Heft: 11 Artikelnummer: 21 Start- / Endseite: - Identifikator: ISSN: 1471-2121