HEMA 3 Staining: A Simple Alternative for the Assessment of Myoblast Differentiation

Abstract

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