Isolation of skeletal muscle stem cells by fluorescence-activated cell sorting. 2015

Ling Liu, and Tom H Cheung, and Gregory W Charville, and Thomas A Rando
The Glenn Center for the Biology of Aging and the Department of Neurology and Neurological Sciences, Stanford University School of Medicine, Stanford, California, USA.

The prospective isolation of purified stem cell populations has dramatically altered the field of stem cell biology, and it has been a major focus of research across tissues in different organisms. Muscle stem cells (MuSCs) are now among the most intensely studied stem cell populations in mammalian systems, and the prospective isolation of these cells has allowed cellular and molecular characterizations that were not dreamed of a decade ago. In this protocol, we describe how to isolate MuSCs from limb muscles of adult mice by fluorescence-activated cell sorting (FACS). We provide a detailed description of the physical and enzymatic dissociation of mononucleated cells from limb muscles, a procedure that is essential in order to maximize cell yield. We also describe a FACS-based method that is used subsequently to obtain highly pure populations of either quiescent or activated MuSCs (VCAM(+)CD31(-)CD45(-)Sca1(-)). The isolation process takes ∼5-6 h to complete. The protocol also allows for the isolation of endothelial cells, hematopoietic cells and mesenchymal stem cells from muscle tissue.

UI MeSH Term Description Entries
D007963 Leukocytes, Mononuclear Mature LYMPHOCYTES and MONOCYTES transported by the blood to the body's extravascular space. They are morphologically distinguishable from mature granulocytic leukocytes by their large, non-lobed nuclei and lack of coarse, heavily stained cytoplasmic granules. Mononuclear Leukocyte,Mononuclear Leukocytes,PBMC Peripheral Blood Mononuclear Cells,Peripheral Blood Human Mononuclear Cells,Peripheral Blood Mononuclear Cell,Peripheral Blood Mononuclear Cells,Leukocyte, Mononuclear
D003584 Cytological Techniques Methods used to study CELLS. Cytologic Technics,Cytological Technic,Cytological Technics,Cytological Technique,Technic, Cytological,Technics, Cytological,Technique, Cytological,Techniques, Cytological,Cytologic Technic,Technic, Cytologic,Technics, Cytologic
D005434 Flow Cytometry Technique using an instrument system for making, processing, and displaying one or more measurements on individual cells obtained from a cell suspension. Cells are usually stained with one or more fluorescent dyes specific to cell components of interest, e.g., DNA, and fluorescence of each cell is measured as it rapidly transverses the excitation beam (laser or mercury arc lamp). Fluorescence provides a quantitative measure of various biochemical and biophysical properties of the cell, as well as a basis for cell sorting. Other measurable optical parameters include light absorption and light scattering, the latter being applicable to the measurement of cell size, shape, density, granularity, and stain uptake. Cytofluorometry, Flow,Cytometry, Flow,Flow Microfluorimetry,Fluorescence-Activated Cell Sorting,Microfluorometry, Flow,Cell Sorting, Fluorescence-Activated,Cell Sortings, Fluorescence-Activated,Cytofluorometries, Flow,Cytometries, Flow,Flow Cytofluorometries,Flow Cytofluorometry,Flow Cytometries,Flow Microfluorometries,Flow Microfluorometry,Fluorescence Activated Cell Sorting,Fluorescence-Activated Cell Sortings,Microfluorimetry, Flow,Microfluorometries, Flow,Sorting, Fluorescence-Activated Cell,Sortings, Fluorescence-Activated Cell
D005819 Genetic Markers A phenotypically recognizable genetic trait which can be used to identify a genetic locus, a linkage group, or a recombination event. Chromosome Markers,DNA Markers,Markers, DNA,Markers, Genetic,Genetic Marker,Marker, Genetic,Chromosome Marker,DNA Marker,Marker, Chromosome,Marker, DNA,Markers, Chromosome
D006614 Hindlimb Either of two extremities of four-footed non-primate land animals. It usually consists of a FEMUR; TIBIA; and FIBULA; tarsals; METATARSALS; and TOES. (From Storer et al., General Zoology, 6th ed, p73) Hindlimbs
D000818 Animals Unicellular or multicellular, heterotrophic organisms, that have sensation and the power of voluntary movement. Under the older five kingdom paradigm, Animalia was one of the kingdoms. Under the modern three domain model, Animalia represents one of the many groups in the domain EUKARYOTA. Animal,Metazoa,Animalia
D013234 Stem Cells Relatively undifferentiated cells that retain the ability to divide and proliferate throughout postnatal life to provide progenitor cells that can differentiate into specialized cells. Colony-Forming Units,Mother Cells,Progenitor Cells,Colony-Forming Unit,Cell, Mother,Cell, Progenitor,Cell, Stem,Cells, Mother,Cells, Progenitor,Cells, Stem,Colony Forming Unit,Colony Forming Units,Mother Cell,Progenitor Cell,Stem Cell
D051379 Mice The common name for the genus Mus. Mice, House,Mus,Mus musculus,Mice, Laboratory,Mouse,Mouse, House,Mouse, Laboratory,Mouse, Swiss,Mus domesticus,Mus musculus domesticus,Swiss Mice,House Mice,House Mouse,Laboratory Mice,Laboratory Mouse,Mice, Swiss,Swiss Mouse,domesticus, Mus musculus
D018482 Muscle, Skeletal A subtype of striated muscle, attached by TENDONS to the SKELETON. Skeletal muscles are innervated and their movement can be consciously controlled. They are also called voluntary muscles. Anterior Tibial Muscle,Gastrocnemius Muscle,Muscle, Voluntary,Plantaris Muscle,Skeletal Muscle,Soleus Muscle,Muscle, Anterior Tibial,Muscle, Gastrocnemius,Muscle, Plantaris,Muscle, Soleus,Muscles, Skeletal,Muscles, Voluntary,Skeletal Muscles,Tibial Muscle, Anterior,Voluntary Muscle,Voluntary Muscles

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