Abstract
A number of chemical mediators regulate neutrophil recruitment to inflammatory sites either positively or negatively. Although the actions of each chemical mediator on the intracellular signaling networks controlling cell migration have been studied with neutrophils cultured in vitro, how such chemical mediators act cooperatively or counteractively in vivo remains largely unknown. To understand the mechanisms regulating neutrophil recruitment to the inflamed intestine in vivo, we recently generated transgenic mice expressing biosensors based on FRET (Förster resonance energy transfer) and set up two-photon excitation microscopy to observe the gastrointestinal tract in living mice. By measuring FRET in neutrophils, we showed activity changes of protein kinases in the neutrophils recruited to inflamed intestines. In this chapter, we describe the protocol used to visualize the protein kinase activities in neutrophils of the inflamed intestine of transgenic mice expressing the FRET biosensors.
Key words
- FRET
- Two-photon microscopy
- Intravital imaging
- Neutrophil recruitment
- Intestinal inflammation
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Acknowledgements
This work was supported by the Platform for Dynamic Approaches to Living System from the Ministry of Education, Culture, Sports, Science and Technology, Japan. We thank K. Otani for assisting with the jugular vein cannulation.
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Mizuno, R., Kamioka, Y., Sakai, Y., Matsuda, M. (2016). Visualization of Signaling Molecules During Neutrophil Recruitment in Transgenic Mice Expressing FRET Biosensors. In: Ivanov, A. (eds) Gastrointestinal Physiology and Diseases. Methods in Molecular Biology, vol 1422. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3603-8_14
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DOI: https://doi.org/10.1007/978-1-4939-3603-8_14
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