Isolation, Purification, and Characterization of Functional Mitochondria Derived from Mouse Skeletal Muscle for...

Mitochondria are essential organelles that regulate energy metabolism, signal transduction, and cellular homeostasis in eukaryotic cells. Mitochondrial dysfunction contributes to the pathogenesis of numerous diseases and has prompted the development of mitochondrial transplantation as a regenerative therapeutic strategy. The successful application of mitochondrial transplantation depends on the availability of highly purified and functionally intact mitochondria. Skeletal muscle is a suitable donor source due to its high mitochondrial content, metabolic activity, and accessibility. This study established a standardized, reproducible protocol for the isolation, purification, and characterization of functional mitochondria from mouse skeletal muscle and evaluated their use in mitochondrial transplantation. The procedure consisted of two major stages. First, mitochondria were isolated from the skeletal muscle of C57BL/6 mice using trypsin digestion followed by differential centrifugation. Second, the isolated mitochondria were characterized to evaluate purity, ultrastructure, and functional activity. Mitochondrial purity was assessed by bicinchoninic acid (BCA) protein quantification and Western blot analysis. Ultrastructural integrity was examined by transmission electron microscopy. Functional activity was evaluated using JC-1 and mitochondrial fluorescent labeling together with measurements of oxygen consumption, ATP production capacity, and respiratory control ratio using a high-resolution respirometry system. The isolated mitochondria exhibited preserved membrane potential, intact ultrastructure, and stable respiratory activity, indicating suitability for downstream functional studies and mitochondrial transplantation applications.