title: Intravital Imaging of Neutrophil Priming Using IL-1β Promoter-driven DsRed Reporter Mice.
abstract: Neutrophils are the most abundant leukocytes in human blood circulation and are quickly recruited to inflammatory sites. Priming is a critical event that enhances the phagocytic functionality of neutrophils. Although extensive studies have unveiled the existence and importance of neutrophil priming during infection and injury, means of visualizing this process in vivo have been unavailable. The protocol provided enables monitoring of the dynamic process of neutrophil priming in living animals by combining three methodologies: 1) DsRed reporter signal - used as a measure of priming 2) in vivo neutrophil labeling - achieved by injection of fluorescence-conjugated anti-lymphocyte antigen 6G (Ly6G) monoclonal antibody (mAb) and 3) intravital confocal imaging. Several critical steps are involved in this protocol: oxazolone-induced mouse ear skin inflammation, appropriate sedation of animals, repeated injections of anti-Ly6G mAb, and prevention of focus drift during imaging. Although a few limitations have been observed, such as the limit of continuous imaging time (~ 8 hr) in one mouse and the leakage of fluorescein isothiocyanate-dextran from blood vessels in the inflammatory state, this protocol provides a fundamental framework for intravital imaging of primed neutrophil behavior and function, which can easily be expanded to examination of other immune cells in mouse inflammation models.
authors.affiliation: Department of Medical Microbiology and Immunology, University of Toledo College of Medicine and Life Sciences; Department of Internal Medicine, Yale University School of Medicine; yyao1@hfhs.org.
authors.forename: Yi
authors.initials: Y
authors.lastname: Yao
authors.affiliation: Department of Medical Microbiology and Immunology, University of Toledo College of Medicine and Life Sciences; Department of Pathophysiology, Southern Medical University (China).
authors.forename: Yun
authors.initials: Y
authors.lastname: Liu
authors.affiliation: Department of Medical Microbiology and Immunology, University of Toledo College of Medicine and Life Sciences.
authors.forename: Akira
authors.initials: A
authors.lastname: Takashima
mesh_terms: D000818:Animals; D053583:Interleukin-1beta; D000069416:Intravital Microscopy; D051379:Mice; D009504:Neutrophils; D011401:Promoter Regions, Genetic
chemical_list: D053583:Interleukin-1beta