Skeletal Muscle Cell Isolation

Skeletal muscle from both hindlimbs was carefully dissected and then gently torn with tissue forceps until homogeneous. Collagenase type 2 (Sigma; 2ml of 2.5 U ml⁻¹), in 10 mM CaCl₂, was added to every two hindlimbs, and the preparation was placed at 37 C for 30 min. After washing, a second enzymatic digestion was performed with Collagenase D (Roche Biochemicals; 1.5 U ml⁻¹) and Dispase II (Roche Biochemicals; 2.4 U ml⁻¹), in a total volume of 1 ml per mouse, at 37 C for 60 min. Preparations were passed through a 40-μm cell strainer (Becton Dickenson), and washed. Resulting single cells were collected by centrifugation at 400g for 5 min.