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

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.