Isolation and Biophysical Characterization of Extracellular Vesicles Released by Myocytes

Extracellular vesicles (EVs) are lipid bilayer-enclosed vesicles released by diverse cell types and found in various body fluids. Because their composition and cargo dynamically respond to physiological and environmental cues, EVs hold promise both as biomarkers and as carriers for therapeutic delivery. Skeletal muscle functions as an endocrine organ, secreting myokines and EVs that modulate a wide range of cellular processes. The murine C2C12 cell line is a widely used in vitro model for investigating muscle biology. Here, we describe a protocol for isolating EVs from differentiated C2C12 myocytes. The isolated EVs are characterized and validated using western blotting, transmission electron microscopy (TEM), and dynamic light scattering (DLS) analysis. This workflow provides a robust platform for studying the molecular composition and functional roles of muscle-derived EVs. Key features • Standardized protocol for isolating extracellular vesicles (EVs) from differentiated C2C12 myocytes. • High-purity EV isolation achieved through sequential ultracentrifugation and size-exclusion chromatography. • Generates EV samples compatible with diverse downstream applications, including western blotting, transmission electron microscopy (TEM), and dynamic light scattering (DLS) analysis. • Enables comparative studies of muscle-derived EV composition and functional changes under both control and treatment conditions in C2C12 myocytes.