Advanced Methodology for Rapid Isolation of Single Myofibers from Flexor Digitorum Brevis Muscle

This paper introduces a refined method for isolating single muscle fibers (myofibers) from the flexor digitorum brevis (FDB) muscle of mice, improving upon existing techniques to achieve higher yields of intact, healthy myofibers. The key improvements include separating the FDB muscle into individual bundles before enzymatic digestion and optimizing the digestion medium, resulting in a more efficient and reproducible process applicable to both young and aged mice. This method aims to accelerate skeletal muscle research by providing a reliable way to obtain functional myofibers for ex vivo studies, potentially reducing the number of animals needed and enabling better screening of muscle disease therapeutics and regeneration strategies.

• 1
  The modified protocol significantly improves the yield of viable, intact myofibers compared to previous methods, achieving approximately 2300–3100 myofibers from two FDB muscles of a single mouse.
• 2
  The optimized digestion times differ between young (35–45 minutes) and aged (80–90 minutes) mice, reflecting age-related changes in muscle tissue, such as increased fibrosis in aged muscle.
• 3
  Functional assessments, including MHC and desmin staining, membrane integrity tests, and calcium transient measurements, confirm that the isolated myofibers are healthy, possess intact sarcolemma, and maintain functional excitation-contraction coupling machinery.