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          Institute: MPI für Infektionsbiologie     Collection: Department of Molecular Biology     Display Documents

ID: 533652.0, MPI für Infektionsbiologie / Department of Molecular Biology
High-throughput and single-cell imaging of NF-kappa B oscillations using monoclonal cell lines
Authors:Bartfeld, Sina; Hess, Simone; Bauer, Bianca; Machuy, Nikolaus; Ogilvie, Lesley A.; Schuchhardt, Johannes; Meyer, Thomas F.
Date of Publication (YYYY-MM-DD):2010-03-16
Title of Journal:BMC Cell Biology
Journal Abbrev.:BMC Cell Biol.
Sequence Number of Article:21
Copyright:© 2010 Bartfeld et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Review Status:Peer-review
Audience:Experts Only
Abstract / Description: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.
Comment of the Author/Creator:This work was supported by funding under the Sixth Research Framework Programme of the European Union, Project INCA (LSHC-CT-2005-018704) and by the BMBF through the RNAi-Net (Grant No. 0313938A) to TFM.
External Publication Status:published
Document Type:Article
Communicated by:Hilmar Fünning
Affiliations:MPI für Infektionsbiologie/Department of Molecular Biology
External Affiliations:MicroDiscovery GmbH, Berlin, Germany.; Hannover Med Sch MHH, D-30625 Hannover, Germany.
Identifiers:ISI:000276333600001 [ID No:1]
ISSN:1471-2121 [ID No:2]
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