Isolation, cultivation and immunostaining of single myofibers: An improved approach to study the behavior of satellite cells


  • Chai Ling Lim Universiti Putra Malaysia
  • King-Hwa Ling Universiti Putra Malaysia
  • Pike-See Cheah Universiti Putra Malaysia



adult stem cell, myofibers, myogenic differentiation 1, paired box family of transcription factors, satellite cell


Satellite cells are myogenic cells responsible for muscle growth shortly after birth and muscle repair/regeneration during adulthood. Therapies based on satellite cells hold promise for treating muscular dysfunctions. Studying satellite cells is technically challenging owing to their low abundance, small size and anatomical dispersed location between the basal lamina and the sarcolemma of myofibers. In this article, we present three improved protocol strategies for studying the properties of satellite cells of the mouse during the different stages of muscle regeneration: (1) immunostaining of freshly isolated single myofibers to facilitate the study of quiescent satellite cells, (2) cultivation of single myofibers on Matrigel®-coated dish to study the myogenesis programs initiated by satellite cell activation, and (3) cultivation of single myofibers in floating conditions to analyze activated satellite cells or the doubling time of satellite cells in myofibers. In brief, when compared to previously published protocols, this article presented an improved protocol that requires shorter experimental time and less laborious approach for higher yield of intact single myofibers for downstream analyses.


Kyle UG, Dupertuis YM, Raguso CA, Hans D, Pichard C. (2003) Appendicular skeletal muscle mass (ASMM) percentiles in 7489 healthy adults aged 18–98 years determined by bioelectrical impedance analysis (BIA). Clin Nutr 22: S1. doi:10.1016/S0261-5614(03)80003-1.

Studitsky AN. (2006) Free auto- and homografts of muscle tissue in experiments on animals. Ann N Y Acad Sci 120: 789-801. doi:10.1111/j.1749-6632.1965.tb30703.x.

Boldrin L, Morgan JE. (2012) Human satellite cells: identification on human muscle fibres. PLoS Curr 3: RRN1294. doi:10.1371/currents.RRN1294.

Yin H, Price F, Rudnicki M. (2013) Satellite Cells and the Muscle Stem Cell Niche. Physiol Rev 93: 23-67. doi:10.1152/physrev.00043.2011.

Bischoff R. (1975) Regeneration of single skeletal muscle fibers in vitro. Anat Rec 182: 215-235. doi:10.1002/ar.1091820207.

Konigsberg UR, Lipton BH, Konigsberg IR. (1975) The regenerative response of single mature muscle fibers isolated in vitro. Dev Biol 45: 260-275. doi:10.1016/0012-1606(75)90065-2.

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. doi:10.1007/bf02634119.

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. doi: 10.1007/978-1-62703-128-8_28.

Anderson JE, Wozniak AC, Mizunoya W. (2012) Single muscle-fiber isolation and culture for cellular, molecular, pharmacological, and evolutionary studies. Methods Mol Biol 798: 85-102. doi: 10.1007/978-1-61779-343-1_6.

Verma M, Asakura A. (2011) Efficient single muscle fiber isolation from alcohol-fixed adult muscle following β-galactosidase staining for satellite cell detection. J Histochem Cytochem 59: 60-67. doi: 10.1369/jhc.2010.956730.

Pasut A, Jones AE, Rudnicki MA. (2013) Isolation and culture of individual myofibers and their satellite cells from adult skeletal muscle. J Vis Exp 22: e50074. doi: 10.3791/50074.

Beauchamp JR, Heslop L, Yu DS, Tajbakhsh S, Kelly RG, et al. (2000) Expression of CD34 and Myf5 defines the majority of quiescent adult skeletal muscle satellite cells. J Cell Biol 151: 1221-1234. doi:10.1083/jcb.151.6.1221.

Gnocchi VF, White RB, Ono Y, Ellis JA, Zammit PS. (2009) Further characterisation of the molecular signature of quiescent and activated mouse muscle satellite cells. PLoS One 4:e 5205. doi: 10.1371/journal.pone.0005205.

Gharaibeh B, Lu A, Tebbets J, Zheng B, Feduska J, et al. (2008) Isolation of a slowly adhering cell fraction containing stem cells from murine skeletal muscle by the preplate technique. Nat Protoc 3: 1501-1509. doi: 10.1038/nprot.2008.142.

Fukada S, Uezumi A, Ikemoto M, Masuda S, Segawa M, et al. (2007) Molecular signature of quiescent satellite cells in adult skeletal muscle. Stem Cells 25: 2448-2459.




How to Cite

Lim CL, Ling K-H, Cheah P-S. Isolation, cultivation and immunostaining of single myofibers: An improved approach to study the behavior of satellite cells. J Biol Methods [Internet]. 2018Mar.16 [cited 2021Oct.21];5(1):e87. Available from: