Single Fiber Isolation Assay for the Assessment of Oxidative Myofiber Behavior

Skeletal muscle is composed of multinucleated myofibers whose integrity and function are essential for movement and overall health. In neuromuscular disorders, muscle groups can be affected differentially across disease types and stages, making it important to isolate viable single fibers from representative muscles for mechanistic studies. The objective of this protocol is to provide a reliable method for isolating a high yield of intact single myofibers from the murine soleus (SOL) muscle, which is challenging to dissociate due to fiber length and fragility. By optimizing collagenase concentration and digestion time, the protocol minimizes fiber loss and preserves viability during dissociation. The optimized workflow markedly reduces residual tissue attachment to isolated fibers, thereby limiting carryover of non-fiber cells and minimizing cross-cell contamination. This technique is suitable for single-fiber analyses as well as satellite cell isolation and subsequent expansion under conditions with minimal contamination. Using this optimized workflow, we routinely obtain ~300-500 intact SOL-derived single myofibers that can be immunolabelled with high efficiency and also yield a high-quality satellite cell population with minimal fibroblasts contamination. Using comparative analyses with Extensor Digitorum Longus (EDL) muscle we underscore the value of a tailored, muscle-specific approach to evaluating therapies in neuromuscular disease research, enabling candidate pharmaceutical interventions to be tested within small, tunable experimental windows.