Satellite cell isolation kit

For magnetic activated cell sorting (MACS), skeletal muscles were dissociated in F10+ media (F10 supplemented with 10% horse serum, and 1% HEPES) containing 0.2% Collagenase II and 0.4% Dispase using the Gentle MACS tissue dissociator for 30–60 min at 37 °C. Single cell suspension was spun down at 500 g for 20 min at 4 °C, washed with F10+ media and filtered using Smart Filters (Miltenyi Biotec). Cells were resuspended in ice-cold PBS 0.5% BSA prior to filtration through a FACS tube cap. SCs were then freshly isolated using first the Satellite Cell Isolation Kit (Miltenyi Biotec) for negative selection, followed by positive selection using α7Integrin beads following the manufacturer’s protocols.
SC cultures and related experiments were performed as previously described³⁴ . Briefly, 5000 SCs (72 h cultures) or 10000 SCs (differentiation assay) were plated on 0.5% ECM in growing media (GM; DMEM, 10%horse serum, FGF2, 1% pen/strep, 1% HEPES). GM was changed every 48 h and differentiation media (DM, DMEM, 2% horse serum, 1% pen/strp, 1% HEPES) was added at 96 h for 18–24 h, monitoring the formation of long and healthy myotubes. All cells were incubated at 37 °C in 5% CO₂ and humid air conditions.

Complete hind limb musculature was minced and incubated with collagenase B (2.5 g/ml, Roche) and dispase II (1 g/ml, Roche) for 30 min at 37 °C. The digested muscles were further homogenized by trituration and filtered through 74-μm cell filters. Isolated cells were pelleted at 450×g and gently resuspended in 500 μl Magnetic-activated cell sorting (MACS) buffer (0.5% BSA, 2 mM EDTA in PBS) followed by addition of 60-μl microbeads coupled to monoclonal antibodies against non-target cells according to instructions provided by the manufacturer (Satellite Cell Isolation Kit, Miltenyi Biotec GmbH). After incubation for 30 min on ice, cells were filtered through a 50-μm filter and loaded on a MACS column (Miltenyi Biotec). The flow-through was collected, washed, and plated on collagen-coated 10-cm cell culture dishes. Staining for Pax7 was performed to check for purity of the isolated cells.

Experimental animals (six-week-old C57BL/6J mice or four-week-old SD rats) were anesthetized with isoflurane and sacrificed by exsanguination. Connective tissue, blood vessels, and fat were carefully removed from muscle collected from the lower limbs using forceps under a stereomicroscope. The collected muscle tissue samples were placed in Hank's balanced salt solution (HBSS-, FUJIFILM Wako Pure Chemical Corporation) containing 1% penicillin-streptomycin and gently shaken to avoid contamination. Myoblast-containing cell suspensions were prepared using the MACS skeletal muscle dissociation kit for mouse and rat (Miltenyi Biotec, North Rhine, Germany). Control mouse myoblasts were isolated by MACS with a purity of 98.5 ± 0.208% (n = 3), using the satellite cell isolation kit (Miltenyi Biotec, North Rhine, Germany), according to the manufacturers' protocols.

Mononucleated cells were obtained from hindlimb muscles of mdx mice aged 3 or 8 weeks, sacrificed by cervical dislocation. The muscles were collected then washed in Ca²⁺- and Mg²⁺-free PBS, minced with scissors, and dissociated enzymatically with type I collagenase (Sigma Aldrich, St. Louis, MO, USA, 2 mg/mL) plus dispase type II (Roche, Basel, Switzerland, 100 mg/mL) in MEM (Gibco, Thermo Fisher Scientific, #11095-080) for 90–120 min in a shaking bath. Enzymes were inactivated adding Hanks’ Balanced Salt Solution (Gibco). Cells were filtered using nylon strainers with decreasing size (100 μm, 70 μm, 40 μm; Falcon, Thermo Fisher Scientific), centrifuged at 300× g for 5 min at 4 °C and counted. Satellite cells were then sorted using satellite Cell Isolation Kit (Milteny Biotec, Bergisch Gladbach, Germany), according to manufacturer’s instructions.

SCs, the stem cells of skeletal muscle, were extracted from the TA muscles of the mice. As previously described, the mouse's TA muscles were dissected under sterile conditions and minced after the blood vessels, connective tissues, and adipogenic tissues were removed [34 (link), 35 ], and then stirred in digestion buffer (0.7% collagenase II in 10% fetal bovine serum [FBS, Gibco, 10099-141, USA) in Dulbecco’s modified Eagle’s medium (DMEM)] at 37 °C for 30 min, followed by centrifugation and filtration [36 ]. A single-cell suspension was prepared and purified using magnetic-activated cell sorting with a Satellite Cell Isolation Kit (Miltenyi Biotec™, 130-104-268, Germany) according to the manufacturer’s instructions [36 ]. Extracted SCs were cultured with myoblast growth medium [DMEM/F12 (Gibco, DMEM-F12, USA) supplemented with 10% FBS and 5 ng/mL fibroblast growth factor 2 (GLPBIO, GP20218, USA)] [37 ]. PAX7, MyoD, MyoG, and Myf5 were used to analyze the state of SCs. Pax7(+) nuclei were considered to be SCs, whereas MyoD(+) Pax7(+) nuclei were considered to be activated for myogenic differentiation, and Myf5(−) Pax7(+) nuclei were considered to be self-renewing SCs.

Hind limb muscles from two tamoxifen-treated Pax7^CreER/+-Rosa26^{dCas9-SunTag/+} mice, aged 6 months, were collected and digested as described above. Satellite cells from digested tissue were isolated using the Satellite Cell Isolation Kit from Miltenyi Biotec, following the manufacturer’s protocol.