Isolation, Culture, and Immunostaining of Skeletal Muscle Myofibers from Wildtype and Nestin-GFP Mice as a Means to Analyze Satellite Cells

摘要: Multinucleated myofibers, the functional contractile units of adult skeletal muscle, harbor mononuclear Pax7+ myogenic progenitors on their surface between myofiber basal lamina and plasmalemma. These progenitors, known as satellite cells, are primary stem cells in muscle. This chapter describes our laboratory protocols for isolating, culturing, immunostaining intact myofibers from mouse muscle a means studying cell dynamics. The first protocol discusses isolation flexor digitorum brevis (FDB) short plated dishes coated with PureCol collagen (formerly Vitrogen) maintained mitogen-poor medium (± supplemental growth factors). Employing such conditions, remain at parent while synchronously undergoing limited number proliferative cycles rapidly differentiate. second longer extensor longus (EDL) EDL routinely individually adherent wells Matrigel mitogen-rich medium, conditions which migrate away myofiber, proliferate extensively, generate numerous differentiating progeny. Alternatively, these can be non-adherent uncoated factors), that retain progeny niche similar to FDB cultures. However, format is preferred choice monitoring freshly isolated (Time 0) myofibers. We conclude this by promoting Nestin-GFP transgenic an efficient tool direct analysis While have been often detected expression Pax7 protein or Myf5nLacZ knockin reporter (approaches also detailed herein), distinctively permits quantification live enables linking initial Time 0 numbers subsequent performance upon culturing. additionally point out implementation transgene other selective lineages illustrated GFP capillaries, endothelial tubes neuronal cells. Myofibers types muscles, diaphragm, masseter, extraocular, analyzed using described herein. Collectively, provides essential tools native position interplay myofiber.