Isolation and culture of skeletal muscle myofibers as a means to analyze satellite cells

Methods in Molecular Biology
Gabi Shefer, Zipora Yablonka-Reuveni

Abstract

Myofibers are the functional contractile units of skeletal muscle. Mononuclear satellite cells located between the basal lamina and the plasmalemma of the myofiber are the primary source of myogenic precursor cells in postnatal muscle. This chapter describes protocols used in our laboratory for isolation, culturing, and immunostaining of single myofibers from mouse skeletal muscle. The isolated myofibers are intact and retain their associated satellite cells underneath the basal lamina. The first protocol discusses myofiber isolation from the flexor digitorum brevis (FDB) muscle. Myofibers are cultured in dishes coated with Vitrogen collagen, and satellite cells remain associated with the myofibers undergoing proliferation and differentiation on the myofiber surface. The second protocol discusses the isolation of longer myofibers from the extensor digitorum longus (EDL). Different from the FDB myofibers, the longer EDL myofibers tend to tangle and break when cultured together; therefore, EDL myofibers are cultured individually. These myofibers are cultured in dishes coated with Matrigel. The satellite cells initially remain associated with the myofiber and later migrate away to its vicinity, resulting in extensive cell prolifer...Continue Reading

Citations

Jun 16, 2017·Current Stem Cell Reports·Veronica PiniHelen C O'Neill
Sep 25, 2004·Developmental Dynamics : an Official Publication of the American Association of Anatomists·Orna HalevyZipora Yablonka-Reuveni
Mar 19, 2021·Frontiers in Cell and Developmental Biology·Yasuro FuruichiYasuko Manabe
Oct 7, 2004·Journal of Cell Science·Gabi SheferZipora Yablonka-Reuveni
May 19, 2021·Skeletal Muscle·Rohan X VermaMarc K Halushka

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