Repair of adult skeletal muscle depends on satellite cells, myogenic stem cells located between the basal lamina and the plasmalemma of the myofiber. Standardized protocols for the isolation and culture of satellite cells are key tools for understanding cell autonomous and extrinsic factors that regulate their performance. Knowledge gained from such studies can contribute important insights to developing strategies for the improvement of muscle repair following trauma and in muscle wasting disorders. This chapter provides an introduction to satellite cell biology and further describes the basic protocol used in our laboratory to isolate and culture satellite cells from adult skeletal muscle. The cell culture conditions detailed herein support proliferation and differentiation of satellite cell progeny and the development of reserve cells, which are thought to reflect the in vivo self-renewal ability of satellite cells. Additionally, this chapter describes our standard immunostaining protocol that allows the characterization of satellite cell progeny by the temporal expression of characteristic transcription factors and structural proteins associated with different stages of myogenic progression. Although emphasis is given here to the isolation and characterization of satellite cells from mouse hindlimb muscles, the protocols are suitable for other muscle types (such as diaphragm and extraocular muscles) and for muscles from other species, including chicken and rat. Altogether, the basic protocols described are straightforward and facilitate the study of diverse aspects of skeletal muscle stem cells.
- Skeletal muscle
- Satellite cell
- Stem cell
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We thank Lindsey Muir for reviewing this manuscript and providing valuable comments. We are also grateful to the granting agencies that funded this study. Our current research is supported by grants to Z.Y.R. from the National Institutes of Health (AG021566; AG035377; AR057794) and the Muscular Dystrophy Association (135908). M.E.D is supported by the Genetic Approaches to Aging Training Program (T32 AG000057). The development of the protocols described here could not be possible without early support to Z.Y.R from the American Heart Association, the USDA Cooperative State Research, Education and Extension Service, and the National Institutes of Health (AG013798).
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Danoviz, M.E., Yablonka-Reuveni, Z. (2012). Skeletal Muscle Satellite Cells: Background and Methods for Isolation and Analysis in a Primary Culture System. In: DiMario, J. (eds) Myogenesis. Methods in Molecular Biology, vol 798. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-343-1_2
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