Isolation and Culture of Individual Myofibers and Their Adjacent Muscle Stem Cells from Aged and Adult Skeletal Muscle.

The isolation and culture of single floating myofibers with their adjacent muscle stem cells allow the analysis and comparison of muscle stem cells from aged and young mice. This method has the advantage that muscle stem cells are cultured on the myofiber, thereby culturing them in conditions as close to their endogenous niche as possible. Here we describe the isolation, culture, transfection with siRNA, and subsequent immunostaining for muscle stem cells on their adjacent myofibers from aged and young mice.

New multiple labelling method for improved satellite cell identification in human muscle: application to a cohort of power-lifters and sedentary men.

Presently applied methods to identify and quantify human satellite cells (SCs) give discrepant results. We introduce a new immunofluorescence method that simultaneously monitors two SC markers (NCAM and Pax7), the basal lamina and nuclei. Biopsies from power-lifters, power-lifters using anabolic substances and untrained subjects were re-examined. Significantly different results from those with staining for NCAM and nuclei were observed. There were three subtypes of SCs; NCAM(+)/Pax7(+) (94%), NCAM(+)/Pax7(-) (4%) and NCAM(-)/Pax7(+) (1%) but large individual variability existed. The proportion of SCs per nuclei within the basal lamina of myofibres (SC/N) was similar for all groups reflecting a balance between the number of SCs and myonuclei to maintain homeostasis. We emphasise that it is important to quantify both SC/N and the number of SCs per fibre. Our multiple marker method is more reliable for SC identification and quantification and can be used to evaluate other markers of muscle progenitor cells.