Isolation of Skeletal Muscle Satellite Cells for <em>In Vitro</em> Myogenesis Studies

Skeletal muscle regeneration and growth depend on satellite cells, the muscle tissue's resident stem cells. Currently, these cells serve as the in vitro model that most accurately represents natural myogenic processes. Nonetheless, the isolation of satellite cells is technically challenging as they exist in only small numbers within skeletal muscle and the typically heterogeneous nature of the isolated cell populations. In this study, we describe a standardized method for isolating and enriching primary myoblasts from skeletal muscles of both adult and neonatal mice. The procedure outlines muscle dissection, enzymatic dissociation, sequential filtration, preplating steps to limit non-myogenic cells, and defined culture conditions for establishing satellite cell-derived myoblast cultures. The protocol provides practical guidance on timing, handling, and critical steps for establishing a successful culture. Primary myoblasts generated using this protocol can be used for a variety of downstream applications, including studies of myogenesis, functional analysis, genetic manipulation, live-cell imaging, or drug testing. The cells reliably fuse and mature, making them well-suited for studying myoblast fusion, muscle development, regenerative mechanisms to restore skeletal muscle function, as well as their metabolism.