title: Visualization of Signaling Molecules During Neutrophil Recruitment in Transgenic Mice Expressing FRET Biosensors.
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.
authors.affiliation: Department of Pathology and Biology of Diseases, Graduate School of Medicine, Kyoto University, Kyoto, 606-8501, Japan.|Department of Surgery, Graduate School of Medicine, Kyoto University, Kyoto, 606-8501, Japan.
authors.forename: Rei
authors.initials: R
authors.lastname: Mizuno
authors.affiliation: Department of Pathology and Biology of Diseases, Graduate School of Medicine, Kyoto University, Kyoto, 606-8501, Japan.|Innovative Techno-Hub for Integrated Medical Bio-Imaging, Kyoto University, Kyoto, Japan.
authors.forename: Yuji
authors.initials: Y
authors.lastname: Kamioka
authors.affiliation: Department of Surgery, Graduate School of Medicine, Kyoto University, Kyoto, 606-8501, Japan.
authors.forename: Yoshiharu
authors.initials: Y
authors.lastname: Sakai
authors.affiliation: Department of Pathology and Biology of Diseases, Graduate School of Medicine, Kyoto University, Kyoto, 606-8501, Japan. matsuda.michiyuki.2c@kyoto-u.ac.jp.|Laboratory of Bioimaging and Cell Signaling, Graduate School of Biostudies, Kyoto University, Kyoto, Japan. matsuda.michiyuki.2c@kyoto-u.ac.jp.
authors.forename: Michiyuki
authors.initials: M
authors.lastname: Matsuda
mesh_terms: D000818:Animals; D015374:Biosensing Techniques; D002465:Cell Movement; D031541:Fluorescence Resonance Energy Transfer; D007249:Inflammation / Q000378:metabolism*; D007421:Intestine, Small / Q000276:immunology* / Q000378:metabolism; D051379:Mice; D008822:Mice, Transgenic; D020556:Neutrophil Infiltration; D009504:Neutrophils / Q000378:metabolism*; D011494:Protein Kinases / Q000378:metabolism*
chemical_list: D011494:Protein Kinases
keywords: FRET; Intestinal inflammation; Intravital imaging; Neutrophil recruitment; Two-photon microscopy