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Isolation of a slowly adhering cell fraction containing stem cells from murine skeletal muscle by the preplate technique.

TitleIsolation of a slowly adhering cell fraction containing stem cells from murine skeletal muscle by the preplate technique.
Publication TypeJournal Article
Year of Publication2008
AuthorsGharaibeh B, Lu A, Tebbets J, Zheng B, Feduska J, Crisan M, Péault B, Cummins J, Huard J
JournalNat Protoc
Volume3
Issue9
Pagination1501-9
Date Published2008
ISSN1750-2799
KeywordsAnimals, Cell Adhesion, Cell Separation, Mice, Muscle, Skeletal, Stem Cells
Abstract

This protocol details a procedure, known as the modified preplate technique, which is currently used in our laboratory to isolate muscle cells on the basis of selective adhesion to collagen-coated tissue culture plates. By employing this technique to murine skeletal muscle, we have been able to isolate a rapidly adhering cell (RAC) fraction within the earlier stages of the process, whereas a slowly adhering cell (SAC) fraction containing muscle-derived stem cells is obtained from the later stages of the process. This protocol outlines the methods and materials needed to isolate RAC and SAC populations from murine skeletal muscle. The procedure involves mechanical and enzymatic digestion of skeletal muscle tissue with collagenase XI, dispase and trypsin followed by plating the resultant muscle slurry on collagen type I-coated flasks where the cells adhere at different rates. The entire preplate technique requires 5 d to obtain the final preplate SAC population. Two to three additional days are usually required before this population is properly established. We also detail additional methodologies designed to further enrich the resultant cell population by continuing the modified preplating process on the SAC population. This process is known as replating and requires further time.

DOI10.1038/nprot.2008.142
Alternate JournalNat Protoc
PubMed ID18772878
Grant ListR01-49684 / / PHS HHS / United States
R01HL069368-01A1 / HL / NHLBI NIH HHS / United States
Publication institute
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