Muscle stem cells (MuSCs) lose a large proportion of their characteristics when removed from their niche, hampering the analysis of muscle stem cell functionality. However, the isolation and culture of single floating myofibers with their adjacent muscle stem cells allow the short-term culture and manipulation of muscle stem cells in conditions as close as possible to the endogenous niche. Here, the isolation, culture and transfection with siRNA of muscle stem cells on their adjacent myofibers from young as well as old mice are described.