Skip to main content
SpringerLink
Log in

Fluorescence Imaging of Bone-Resorbing Osteoclasts by Confocal Microscopy

  • Protocol
Bone Research Protocols

Part of the book series: Methods in Molecular Medicine ((MIMM,volume 80))

  • 1768 Accesses

Abstract

Osteoclasts are large multinucleate bone cells with the capacity to degrade bone by the process of bone resorption and, thus, participate in the homeostasis of bone and calcium in the body (1). Imaging of osteoclasts can be performed by a variety of microscopy methods including light microscopy, electron microscopy, and atomic force microscopy (AFM) (2,3). These techniques, together with histochemical and immunocytochemical stains, enable the researcher to analyze the cellular structure and function of this complex cell type both in vivo within bone tissues and isolated in vitro in primary cell cultures (see Part II, Culture of Osteoclasts).

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 119.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 159.00
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Teitelbaum, S. L. (2000) Bone-resorption by osteoclasts. Science 289, 1504–1508.

    Article  PubMed  CAS  Google Scholar 

  2. McKee, M. D. and Nanci, A. (1996) Microscopy of bone. Microsc. Res. Tech. 33, 92–239.

    Article  Google Scholar 

  3. Lehenkari, P., Charras, G., Nesbitt, S., and Horton, M. (2000) New technologies in scanning probe microscopy for studying molecular interactions in cells. Expert Reviews. http://www-ermm.cbcu.cam.ac.uk/00001575h.htm.

  4. Reynaud, K., Nogueira, R., Kurzawa, R., and Smitz, J. (2001) Confocal microscopy: principles and applications to the field of reproductive biology. Folia Histochem. Cytobiol. 39, 75–85.

    PubMed  CAS  Google Scholar 

  5. Lakkakorpi, P. T., Helfrich, M. H., Horton, M. A., and Väänänen, H. K. (1993) Spatial organization of microfilaments and vitronectin receptor, αvβ3, in osteoclasts. J. Cell Sci. 104, 663–670.

    PubMed  CAS  Google Scholar 

  6. Nesbitt, S. A. and Horton, M. A. (1997). Trafficking of matrix collagens through bone-resorbing osteoclasts. Science 276, 266–269.

    Article  PubMed  CAS  Google Scholar 

  7. Nesbitt, S., Charras, G., Lehenkari, P., and Horton, M. (2000) Three-dimensional imaging of bone-resorbing osteoclasts: spatial analysis of matrix collagens, cathepsin K, MMP-9 and TRAP by confocal microscopy. J. Bone Miner. Res. 15, 1219.

    Google Scholar 

  8. Baron, R., Neff, L., Brown, W., Courtoy, P., Louvard, D., and Farquhar, M. (1988) Polarised secretion of lysosomal enzymes: co-distribution of cation-independent mannose-6-phosphate receptors and lysosomal enzymes along the osteoclast exocyotic pathway. J. Cell Biol. 106, 1863–1872.

    Article  PubMed  CAS  Google Scholar 

  9. Horton, M. A, Nesbitt S. A., Bennett, J. H., and Stenbeck, G. (2001) Integrins and other cell surface attachment molecules of bone cells, in Principles of Bone Biology, 2nd edit. (Bilezikian, J. P., Raisz, L. G., and Rodan G. A., eds.). Academic Press, San Diego.

    Google Scholar 

  10. Wucherpfennig, A., Li, Y., Stetler-Steveson, W., Rosenberg, A., and Stashenko P. (1994) Expression of 92 kD type IV collagenase/gelatinase B in human osteoclasts. J. Bone Miner. Res. 9, 549–556.

    Article  PubMed  CAS  Google Scholar 

  11. Nesbitt, S. and Horton, M. (1999) Extracellular annexin II increases trafficking of matrix collagens through bone-resorbing osteoclasts to promote bone resorption. Calcif. Tissue Int. 64(Suppl. 1), S35.

    Google Scholar 

  12. Boyde, A., Ali, N. N., and Jones, S. J. (1984) Resorption of dentine by isolated osteoclasts in vitro. Br. Dent. J. 156, 216–220.

    Article  PubMed  CAS  Google Scholar 

  13. Chambers, T. J., Revell, P. A., Fuller, K., and Athanasou, N. A. (1984) Resorption of bone by isolated rabbit osteoclasts. J. Cell Sci. 66, 383–399.

    PubMed  CAS  Google Scholar 

  14. Arnett, T. D. and Dempster, D. W. (1986) Effect of pH on bone resorption by rat osteoclasts in vitro. Endocrinology 119, 119–124.

    Article  PubMed  CAS  Google Scholar 

  15. Horton, M. A., Rimmer, E. F., Lewis, D., Pringle, J., Fuller, K., and Chambers, T. J. (1984) Cell surface characterisation of the human osteoclast: phenotypic relationship to other bone marrow-derived cell types. J. Pathol. 144, 282.

    Article  Google Scholar 

  16. Atkins, G., Haynes, D., Graves, S., Evdokiou, S., Bouralexis, S., and Findlay, D. (2000) Expression of osteoclast differentiation signals by stromal elements of giant cell tumors. J. Bone Miner. Res. 15, 640–649.

    Article  PubMed  CAS  Google Scholar 

  17. James, I., Lark, M., Zembryki, D., et al. (1999) Development and characterisation of a human in vitro resorption assay: Demonstration of utility using novel antiresorption agents. J. Bone and Miner. Res. 14, 1562–1569.

    Article  CAS  Google Scholar 

  18. Helfrich, M. H., Nesbitt, S. A., Dorey, E. L., and Horton, M. A. (1992). Rat osteoclasts adhere to a wide range of RGD (Arg-Gly-Asp) peptide-containing proteins, including the bone sialoproteins and fibronectin, via a β3 integrin. J. Bone Min. Res. 7, 332–343.

    Google Scholar 

  19. Horton, M. A. (2001) Integrin antagonists as inhibitors of bone resorption: implications for treatment. Proc. Nutr. Soc. 60, 275–281.

    PubMed  CAS  Google Scholar 

  20. Helfrich, M. H., Nesbitt, S. A., Lakkakorpi, P. T., et al. (1996) β1 integrins and osteoclast function: involvement in collagen recognition and bone resorption. Bone 19, 317–328.

    Article  PubMed  CAS  Google Scholar 

  21. Palokangas, H., Mulari, M., and Vannanen, K. (1997) Endocytotic pathway from the basal plasma membrane to the ruffled border membrane in bone-resorbing osteoclasts. J. Cell Sci. 110, 1767–1780.

    PubMed  CAS  Google Scholar 

  22. Chavrier, P., Parton, R. G., Hauri, H. P., Simons, K., and Zerial, M. (1990) Localization of low molecular weight GTP binding proteins to exocytotic and endocytotic compartments. Cell 62, 317–329.

    Article  PubMed  CAS  Google Scholar 

  23. Horton, M. A, Townsend, P. A., and Nesbitt, S. A. (1996) Cell surface attachment molecules in bone, in, Principles of Bone Biology (Bilezikian, J. P., Raisz, L. G., and Rodan, G. A., eds.), pp. 217–230. Academic Press, San Diego.

    Google Scholar 

  24. Guilak, F. (1994) Volume and surface area measurement of viable chondrocytes in situ using geometric modelling of serial confocal sections. J. Microsc. 173, 245–256.

    PubMed  CAS  Google Scholar 

  25. Salo, J., Metsikkö, K., Palokangas, H., Lehenkari, P., and Väänänen, H. K. (1996) Bone-resorbing osteoclasts reveal a dynamic divison of basal plasma membrane into two different domains. J. Cell Sci. 109, 301–307.

    PubMed  CAS  Google Scholar 

  26. Townsend, P. A., Vilanoova, I., Teti, A., and Horton, M. A. (1999) β1 integrin antisense oligodeoxynucleotides: utility in controlling osteoclast function. Eur. J. Cell Biol. 78, 448–496.

    Google Scholar 

  27. Stenbeck, G. and Horton, M. A. (2000) A new specialized cell-matrix interaction in actively resorbing osteoclasts. J. Cell Sci. 113, 1577–1587.

    PubMed  CAS  Google Scholar 

  28. Walsh, C. A., Carron, J. A., and Gallagher, J. A. (1996) Isolation of osteoclasts from human giant cell tumors and long-term marrow cultures, in Methods in Molecular Medicine: Human Cell Culture Protocols (Jones, G. E., ed.), Humana Press, Totowa, NJ.

    Google Scholar 

  29. Nesbitt, S. and Horton, M. (1992) A nonradioactive biochemical characterisation of membrane proteins using enhanced chemiluminescence. Analyt. Biochem. 206, 267–272.

    Article  PubMed  CAS  Google Scholar 

  30. Hnatowitch, D. J., Virzi, F., and Rusckowski, M. (1987) Investigations of avidin and biotin for imaging applications. J. Nucl. Med. 28, 1294–1302.

    Google Scholar 

  31. Silver, I. A., Murrills, R. J., and Etherington, D. J. (1988) Microelectrode studies on the acid microenvironment beneath adherent macrophages and osteoclasts. Exp. Cell. Res. 175, 266–276.

    Article  PubMed  CAS  Google Scholar 

  32. Salo, J., Lehenkari, P., Mulari, M., Metsikkö, K., and Väänänen, H. K. (1997) Removal of osteoclast bone resorption products by transcytosis. Science 276, 270–273.

    Article  PubMed  CAS  Google Scholar 

  33. Lader, C., Scopes, J., Horton, M. and Flanagen, A. (2001) Generation of human osteoclasts in stromal cell-free and stromal cell-rich cultures: differences in osteoclast CD11c/CD18 integrin expression. Br. J. Haematol 112, 430–437.

    Article  PubMed  CAS  Google Scholar 

  34. Squirrell, J., Wokosin, D., White, J., and Bavister, B. (1999) Long-term two photon fluorescence imaging of mammalian embryos without compromising viability. Nat. Biotechnol. 17, 763–767.

    Article  PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Bone and Mineral Centre, The Rayne Institute, Department of Medicine, University College London, London, UK

    Stephen A. Nesbitt & Michael A. Horton

Authors
  1. Stephen A. Nesbitt
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Michael A. Horton
    View author publications

    You can also search for this author in PubMed Google Scholar

Editor information

Editors and Affiliations

  1. University of Aberdeen Medical School, Aberdeen, UK

    Miep H. Helfrich PhD  & Stuart H. Ralston MD  & 

Rights and permissions

Reprints and permissions

Copyright information

© 2003 Humana Press Inc., Totowa, NJ

About this protocol

Cite this protocol

Nesbitt, S.A., Horton, M.A. (2003). Fluorescence Imaging of Bone-Resorbing Osteoclasts by Confocal Microscopy. In: Helfrich, M.H., Ralston, S.H. (eds) Bone Research Protocols. Methods in Molecular Medicine, vol 80. Humana Press. https://doi.org/10.1385/1-59259-366-6:259

Download citation

  • DOI: https://doi.org/10.1385/1-59259-366-6:259

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-58829-044-1

  • Online ISBN: 978-1-59259-366-8

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation