James, Conrad D. and Moorman, Matthew W. and Carson, Bryan D. and Branda, Catherine S. and Lantz, Jeffrey W. and Manginell, Ronald P. and Martino, Anthony and Singh, Anup K. (2009) Nuclear translocation kinetics of NF-kappa B in macrophages challenged with pathogens in a microfluidic platform. Biomedical Microdevices, 11 (3). pp. 693-700.
We have developed a microfluidic platform for real-time imaging of host-pathogen interactions and cellular signaling events. Host cells are immobilized in a controlled environment for optical interrogation of the kinetics and stochasticity of immune response to pathogenic challenges. Here, we have quantitatively measured activation of the toll-like receptor 4 (TLR4) pathway in RAW264.7 murine macrophage-like cells. This was achieved by measuring the cytoplasm-to-nucleus translocation kinetics of a green fluorescent protein fusion construct to the NF-kappa B transcription factor subunit RelA (GFP-RelA). Translocation kinetics in response to live bacteria and purified lipopolysaccharide (LPS) challenges were measured, and this work presents the first demonstration of live imaging of host cell infection on a microfluidic platform with quantitative analysis of an early (< 0.5 h from infection) immune signaling event. Our data show that a 1,000x increase in the LPS dose led to a similar to 10x increase in a host cell activation metric we developed in order to describe NF-kappa B translocation kinetics. Using this metric, live bacteria challenges were assigned an equivalent LPS dose as a first step towards comparing NF-kappa B translocation kinetics between TLR4-only pathway signaling (activated by LPS) and multiple pathway signaling (activated by whole bacteria). The device also contains a unique architecture for capturing and fluidically isolating single host cells for the purpose of differentiating between primary and secondary immune signaling.
|Subjects:||Biomedical Science > Nanobiotechnology|
|Deposited On:||25 Aug 2010 10:21|
|Last Modified:||25 Aug 2010 10:21|
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