title: Use of intravital microscopy and in vitro chemotaxis assays to study the roles of sphingosine-1-phosphate in bone homeostasis.
abstract: We describe a method to visualize the migration of osteoclast precursors within intact murine bone -marrow in real time using intravital multiphoton microscopy. Conventionally, cell migration has been evaluated using in vitro systems, such as transmigration assays. Although these methods are convenient for quantification and are highly reproducible, these in vitro assay systems may not accurately reflect in vivo cellular behavior. In addition to in vitro analyses, recent technological progress in two-photon excitation-based laser microscopy has enabled the visualization of dynamic cell behavior deep inside intact living organs. Combining this imaging method with in vitro chemoattraction analyses, we have revealed that sphingosine-1-phosphate (S1P), a lipid mediator enriched in blood, bidirectionally controls the trafficking of osteoclast precursors between the circulation and bone marrow cavities via G protein-coupled receptors (GPCRs).
authors.affiliation: Laboratory of Cellular Dynamics, WPI-Immunology Frontier Research Center, Osaka University, Suita, Japan.
authors.forename: Taeko
authors.initials: T
authors.lastname: Ishii
authors.forename: Shunsuke
authors.initials: S
authors.lastname: Kawamura
authors.forename: Issei
authors.initials: I
authors.lastname: Nishiyama
authors.forename: Junichi
authors.initials: J
authors.lastname: Kikuta
authors.forename: Masaru
authors.initials: M
authors.lastname: Ishii
mesh_terms: D000818:Animals; D001854:Bone Marrow Cells / Q000166:cytology; D001842:Bone and Bones / Q000166:cytology* / Q000378:metabolism*; D054443:Cell Migration Assays / Q000379:methods*; D002465:Cell Movement; D002633:Chemotaxis; D005260:Female; D006706:Homeostasis; D008246:Lysophospholipids / Q000172:deficiency / Q000378:metabolism*; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D008822:Mice, Transgenic; D036641:Microscopy, Fluorescence, Multiphoton; D010010:Osteoclasts / Q000166:cytology / Q000378:metabolism; D013110:Sphingosine / Q000031:analogs & derivatives* / Q000172:deficiency / Q000378:metabolism
chemical_list: D008246:Lysophospholipids; C060506:sphingosine 1-phosphate; D013110:Sphingosine