Use of Intravital Microscopy and In Vitro Chemotaxis Assays to Study the Roles of Sphingosine-1-Phosphate in Bone Homeostasis

  • Taeko Ishii
  • Shunsuke Kawamura
  • Issei Nishiyama
  • Junichi Kikuta
  • Masaru IshiiEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 874)


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).

Key words

Intravital imaging Multiphoton microscopy Migration Osteoclast precursor S1P S1PR1 S1PR2 



This work was supported by Grants-in-Aid for Encouragement of Young Scientists (A) (22689030), for Scientific Research on Innovative Areas (22113007) and by a Funding Program for World-Leading Innovative R&D on Science and Technology (FIRST Program) from the Ministry of Education, Science, Sports and Culture of Japan, by a Grant-in-Aid for Research on Allergic Disease and Immunology (H21-010) from the Ministry of Health, Labor and Welfare of Japan, and by Grants from the International Human Frontier Science Program (CDA-00059/2009 and RGY-0077/2011).


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Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • Taeko Ishii
    • 1
  • Shunsuke Kawamura
    • 1
  • Issei Nishiyama
    • 1
  • Junichi Kikuta
    • 1
  • Masaru Ishii
    • 1
    Email author
  1. 1.Laboratory of Cellular Dynamics, WPI-Immunology Frontier Research CenterOsaka UniversitySuitaJapan

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