Effects of culture time on muscle fiber morphology and function. (A) Graphical overview of the culture experiment (image made in BioRender). (B) Representative confocal maximum intensity projection images taken with a 60× objective stained for α-actinin (green), myosin (red), and nuclei (blue) in muscle fibers cultured at days 1 and 7. Note the disordered sarcomere structure and spreading of the end of the fiber at day 7. (C) Muscle fiber length at days 1 and 7 of culture. (D) Muscle fiber width at day 1 and day 7 of culture. (E–G): Contractile measurements of muscle fibers kept in culture for 1 and 7 days. (E) Quantification of muscle fiber resting sarcomere length at days 1 and 7 of ex vivo culture. (F) Quantification of the percentage of sarcomere shortening at days 1 and 7 of ex vivo culture. (G) Quantification of the contractile velocity of muscle fiber at days 1 and 7 of ex vivo culture. Data are means ± SEM; large dots represent mean values per mouse, and small dots represent single muscle fibers. Significance was determined using Student’s t test with P < 0.05 considered significant with * = P < 0.05 and ** = P < 0.01.