Skeletal muscle tissue regeneration requires quiescent satellite cell activation, proliferation, and differentiation. Regenerative capacity of satellite cells can be studied in vitro by differentiating under low-serum conditions (2% to 5%) to form multinucleated myotubes. Myotubes are fixed and stained, and indices of differentiation are quantified. Jenner and Giemsa stains are typically used for myotube staining; however, this staining process can be variable depending on factors such as stain pH, staining time, and time since stain preparation. This article includes protocols for myoblast isolation, proliferation, and differentiation in vitro; Jenner-Giemsa staining; HEMA 3 staining; and quantification. Representative images using each staining method and quantification are included. The protocols identify critical steps and considerations for cell culture and each staining method and provide an even simpler alternative to Jenner-Giemsa staining. © 2019 by John Wiley & Sons, Inc. Basic Protocol 1: Primary myoblast isolation Alternate Protocol 1: Plating cryopreserved myoblasts Basic Protocol 2: Myoblast passage and expansion Basic Protocol 3: Myoblast differentiation Basic Protocol 4: HEMA 3 staining Alternate Protocol 2: Jenner-Giemsa staining Basic Protocol 5: Quantification of myotube density Basic Protocol 6: Quantification of fusion index Basic Protocol 7: Quantification of myotubes per field.
Keywords: differentiation index; imaging; myoblast isolation; myotube staining.
© 2019 John Wiley & Sons, Inc.