Skip to main content
SpringerLink
Log in
Book cover

Skeletal Muscle Regeneration in the Mouse pp 141–162Cite as

Isolation, Culture, Functional Assays, and Immunofluorescence of Myofiber-Associated Satellite Cells

  • Protocol
  • First Online:

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1460))

Abstract

Adult skeletal muscle stem cells, termed satellite cells, regenerate and repair the functional contractile cells in adult skeletal muscle called myofibers. Satellite cells reside in a niche between the basal lamina and sarcolemma of myofibers. Isolating single myofibers and their associated satellite cells provides a culture system that partially mimics the in vivo environment. We describe methods for isolating and culturing intact individual myofibers and their associated satellite cells from the mouse extensor digitorum longus muscle. Following dissection and isolation of individual myofibers we provide protocols for myofiber transplantation, satellite cell transfection, immune detection of satellite cell antigens, and assays to examine satellite cell self-renewal and proliferation.

* Authors Thomas O. Vogler and Katherine E.Gadek contributed equally.

This is a preview of subscription content, 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   109.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD   139.00
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD   159.99
Price excludes VAT (USA)
  • Durable hardcover 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

Learn about institutional subscriptions

Springer Nature is developing a new tool to find and evaluate Protocols. Learn more

References

  1. Yin H, Price F, Rudnicki MA (2013) Satellite cells and the muscle stem cell niche. Physiol Rev 93:23–67

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Keefe AC, Lawson JA, Flygare SD, Fox ZD, Colasanto MP, Mathew SJ, Yandell M, Kardon G (2015) Muscle stem cells contribute to myofibres in sedentary adult mice. Nat Commun 6:7087. doi:10.1038/ncomms8087

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Pawlikowski B, Pulliam C, Betta ND, Kardon G, Olwin BB (2015) Pervasive satellite cell contribution to uninjured adult muscle fibers. Skelet Muscle 5:1–13. doi:10.1186/s13395-015-0067-1

    Article  Google Scholar 

  4. Mauro A (1961) Satellite cell of skeletal muscle fibers. J Biophys Biochem Cytol 9:493–495

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Cheung TH, Rando TA (2013) Molecular regulation of stem cell quiescence. Nat Rev Mol Cell Biol 14:329–340. doi:10.1038/nrm3591

    Article  CAS  PubMed  Google Scholar 

  6. Doles JD, Olwin BB (2015) Muscle stem cells on the edge. Curr Opin Genet Dev 34:24–28. doi:10.1016/j.gde.2015.06.006

    Article  CAS  PubMed  Google Scholar 

  7. Gilbert PM, Havenstrite KL, Magnusson KE, Sacco A, Leonardi NA, Kraft P, Nguyen NK, Thrun S, Lutolf MP, Blau HM (2010) Substrate elasticity regulates skeletal muscle stem cell self-renewal in culture. Sci Signal 329:1078

    CAS  Google Scholar 

  8. Chakkalakal JV, Jones KM, Basson MA, Brack AS (2012) The aged niche disrupts muscle stem cell quiescence. Nature 490:355–360. doi:10.1038/nature11438

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Bischoff R (1986) Proliferation of muscle satellite cells on intact myofibers in culture. Dev Biol 115:129–139

    Article  CAS  PubMed  Google Scholar 

  10. Rosenblatt JD, Lunt AI, Parry DJ, Partridge TA (1995) Culturing satellite cells from living single muscle fiber explants. In Vitro Cell Dev Biol Anim 31:773–779

    Article  CAS  PubMed  Google Scholar 

  11. Bernet JD, Doles JD, Hall JK, Kelly Tanaka K, Carter TA, Olwin BB (2014) p38 MAPK signaling underlies a cell-autonomous loss of stem cell self-renewal in skeletal muscle of aged mice. Nat Med 20:265–271. doi:10.1038/nm.3465

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Troy A, Cadwallader AB, Fedorov Y, Tyner K, Tanaka KK, Olwin BB (2012) Coordination of satellite cell activation and self-renewal by Par-complex-dependent asymmetric activation of p38alpha/beta MAPK. Cell Stem Cell 11:541–553. doi:10.1016/j.stem.2012.05.025

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Shefer G, Yablonka-Reuveni Z (2005) Isolation and culture of skeletal muscle myofibers as a means to analyze satellite cells. Methods Mol Biol 290:281–304

    PubMed  PubMed Central  Google Scholar 

  14. Delaurier A, Burton N, Bennett M, Baldock R, Davidson D, Mohun TJ, Logan MP (2008) The Mouse Limb Anatomy Atlas: an interactive 3D tool for studying embryonic limb patterning. BMC Dev Biol 8:83. doi:10.1186/1471-213X-8-83

    Article  PubMed  PubMed Central  Google Scholar 

  15. Keire P, Shearer A, Shefer G, Yablonka-Reuveni Z (2013) Isolation and culture of skeletal muscle myofibers as a means to analyze satellite cells. Methods Mol Biol 946:431–468. doi:10.1007/978-1-62703-128-8_28

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Hall JK, Banks GB, Chamberlain JS, Olwin BB (2010) Prevention of muscle aging by myofiber-associated satellite cell transplantation. Sci Transl Med 2:57ra83. doi:10.1126/scitranslmed.3001081

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Author information

Author notes

    Authors and Affiliations

    1. Department of Molecular, Cellular and Developmental Biology, University of Colorado at Boulder, Boulder, CO, 80309, USA

      Thomas O. Vogler, Katherine E. Gadek, Adam B. Cadwallader, Tiffany L. Elston & Bradley B. Olwin

    Authors
    1. Thomas O. Vogler
      View author publications

      You can also search for this author in PubMed Google Scholar

    2. Katherine E. Gadek
      View author publications

      You can also search for this author in PubMed Google Scholar

    3. Adam B. Cadwallader
      View author publications

      You can also search for this author in PubMed Google Scholar

    4. Tiffany L. Elston
      View author publications

      You can also search for this author in PubMed Google Scholar

    5. Bradley B. Olwin
      View author publications

      You can also search for this author in PubMed Google Scholar

    Corresponding author

    Correspondence to Bradley B. Olwin .

    Editor information

    Editors and Affiliations

    1. Lillehei Heart Institute, University of Minnesota, Minneapolis, Minnesota, USA

      Michael Kyba

    Rights and permissions

    Reprints and permissions

    Copyright information

    © 2016 Springer Science+Business Media New York

    About this protocol

    Cite this protocol

    Vogler, T.O., Gadek, K.E., Cadwallader, A.B., Elston, T.L., Olwin, B.B. (2016). Isolation, Culture, Functional Assays, and Immunofluorescence of Myofiber-Associated Satellite Cells. In: Kyba, M. (eds) Skeletal Muscle Regeneration in the Mouse. Methods in Molecular Biology, vol 1460. Springer, New York, NY. https://doi.org/10.1007/978-1-4939-3810-0_11

    Download citation

    • DOI: https://doi.org/10.1007/978-1-4939-3810-0_11

    • Published:

    • Publisher Name: Springer, New York, NY

    • Print ISBN: 978-1-4939-3808-7

    • Online ISBN: 978-1-4939-3810-0

    • eBook Packages: Springer Protocols

    Publish with us

    Policies and ethics

    Search

    Navigation