Background

Although skeletal muscle is a fully differentiated post-mitotic tissue, it maintains intrinsic capability to regenerate in response to both genetic and acquired forms of muscle fiber damage (Le Grand and Rudnicki, 2007). Muscle regeneration in adults is mediated by a population of stem cells known as satellite cells which reside between the basal lamina and sarcolemma of myofibers in a mitotically quiescent state (Le Grand and Rudnicki, 2007). In response to muscle trauma, satellite cells become activated and proliferate to produce myoblasts that fuse with pre-existing fibers and with one another to repair or produce new myofibers. A small portion of satellite cells does not differentiate but instead reenters quiescence to maintain the stem cell pool. Satellite cells of all mammalian species express paired-box (Pax) transcription factor Pax7 which is also used as a critical marker to determine the fate of satellite cells in association with other myogenic factors such as MyoD (Le Grand and Rudnicki, 2007; Kuang et al., 2006; Kuang and Rudnicki, 2008).

The in vivo process of muscle regeneration with respect to satellite cell activation, proliferation, and differentiation can be partly recapitulated through suspension culture of myofiber explants (Rosenblatt et al., 1995; Shefer and Yablonka-Reuveni, 2005). The process of myofiber isolation which involves digestion of muscle tissue with matrix-degrading enzymes (e.g., collagenases) and mechanical shearing causes minor trauma which leads to the activation of satellite cells on myofibers. Immediately upon isolation, each myofiber is associated with a fixed number (Pax7⁺/MyoD^-) of satellite cells resting in quiescence. At around 24 h in culture, satellite cells undergo their first round of cell division, through upregulating MyoD (Pax7⁺/MyoD⁺) and proliferate to form cell aggregates by 72 h. Satellite cells on cultured myofiber explants can either terminally differentiate (Pax7^-/MyoD⁺) or self-renew (Pax7⁺/MyoD^-) and return to quiescence (Le Grand and Rudnicki, 2007). The myofiber culture system has served as an excellent platform to study the fundamental properties of muscle stem cells and the effects of various genetic manipulations on the basic properties of satellite cells. One of the major advantages of isolated single myofiber cultures is that the physical interaction between the myofiber and satellite cells is preserved in the sense that satellite cells are still maintained beneath the basal lamina (Bischoff, 1986). Furthermore, suspension cultures of myofibers are routinely used to study the effects of various pharmacological compounds and overexpression or knockdown of specific proteins on self-renewal, proliferation, or differentiation of satellite cells (Shefer and Yablonka-Reuveni, 2005; Anderson et al., 2012; Keire et al., 2013). They also provide a useful tool to study the motility of satellite cells on myofibers through live imaging (Siegel et al., 2009). In addition, a few investigators have used isolated single myofibers explants to prepare pure myoblast cultures in which the cells are mostly derived from the myofiber-associated satellite cells.

The isolation of single myofibers from flexor digitorum brevis (FDB) was first described by Bischoff (1986). This protocol was modified later by Rosenblatt et al. to allow handling of single myofibers after collagenase digestion (Rosenblatt et al., 1995). Since then several modifications have been proposed which improved the yield and the handling of the isolated myofibers (Shefer and Yablonka-Reuveni, 2005; Anderson et al., 2012; Verma and Asakura, 2011; Pasut, 2013). However, most of the published protocols still do not produce a sufficient number of myofibers in a single preparation primarily because of under or over digestion with collagenase and the loss of myofibers during picking, sub-culturing, or staining. The amount of time to digest a muscle from rodents may vary with genetic background and healthy versus diseased muscle. In our laboratory, we are now using a six-well tissue culture plate in which the digestion and physical separation of individual myofibers can be visually monitored under a phase contrast microscope. Furthermore, we have developed an improved protocol to stain the myofiber-associated satellite cells for various markers (Hindi et al., 2012; Hindi and Kumar, 2016; Ogura et al., 2013). In this article, we provide the detailed protocol for the isolation, culturing, and staining of myofiber-associated satellite cells for Pax7 and MyoD protein. The same protocol can be adapted for staining of other proteins in satellite cells of myofiber explant.