70 and 40 μm cell strainers

Flow cytometry and cell sorting were carried out as we previously described [14 (link)]. In brief, lungs were isolated in Hank’s Balanced Salt Solution (HBSS, Gibco), minced into small pieces and incubated with 0.5% collagenase type IV in HBSS (Life Technologies) at 37 °C for 45 min. Lung homogenates were then passed through 70 and 40 μm cell strainers (BD Biosciences) to obtain single-cell suspensions. Cells were centrifuged at 4 °C at 1000 rpm for 5 min and then resuspended in MACS buffer and stained with anti-PDGFRα (APC-conjugated, 1:100), anti-EpCAM (APC-Cy7-conjugated, 1:100), anti-CD31 (Pacific Blue-conjugated, 1:100) and anti-CD45 (Pacific Blue-conjugated, 1:100) antibodies (all from Biolegend) for 20 min on ice in the dark. Then, cells were washed with MACS buffer. Flow cytometry and cell sorting were done using FACSAria III cell sorter (BD Biosciences).

Primary muscle cells were derived from hindlimb muscles of two-three month-old mice (two mice for each preparation) as described previously^{41 (link)}. Briefly, muscles were digested with 0.2% collagenase type-II (Sigma) in DMEM for 30 minutes at 37 °C, and then with 2 mg/ml Collagenase/Dispase (Roche Diagnostic) for 30 minutes at 37 °C. Satellite cells were mechanically dissociated by passing the tissue suspension through a 5 ml pipette; the slurry was sequentially filtered through 70 and 40 μm cell strainers (BD Biosciences) and centrifuged. Pelleted cells were resuspended in F-10 (Gibco) supplemented with 20% FBS, 2.5 ng/ml basis-FGF (Peprotech), and penicillin-streptomycin. The cell suspension was preplated for one hour on uncoated dishes to remove fibroblasts. The medium containing the enriched myoblast population was then plated on tissue culture dishes coated with type-I collagen (Sigma). After three days in culture, primary myoblasts were shifted to differentiation medium and harvested for RNA preparation after 72 hours.