Isolation and Culture of Mouse Satellite Cells

Quiescent satellite cells were collected from the skeletal muscles of 8-week-old male SJS-model mice using fluorescence-activated cell sorting and an SM/C-2.6 antibody, as described previously [31 (link)]. The SM/C-2.6 antibody was provided by Dr. Fukada. These satellite cells were seeded on rhLaminin-521 (Thermo Fisher Scientific)-coated plates and cultured in satellite cell growth media (Dulbecco’s modified Eagle’s medium (DMEM), 20% fetal bovine serum, 10% horse serum, chicken embryo extract (United States Biological, Salem, MA, USA), and penicillin/streptomycin) containing 2.5 µg/mL recombinant human FGF (ProteinTech, Rosemont, IL, USA), as previously described [32 (link),33 (link)]. Satellite cells were differentiated to myotubes by culturing in high glucose DMEM (Thermo Fisher Scientific), supplemented with 2% horse serum (Thermo Fisher Scientific) and penicillin/streptomycin. To rescue perlecan-null myotubes, recombinant perlecan protein (20 μg/mL; purified and provided by Dr. Sasaki) was added at the start of the differentiation process. Calcium imaging experiments were performed 12–16 h after adding 70 µg/mL laminin/entactin complex (Corning Inc., Corning, NY, USA) and 10 ng/mL recombinant neural agrin (R&D Systems, Minneapolis, MN, USA).