F 10 ham s medium

As described previously [33 (link)], primary myoblasts were isolated from fore and hind limb skeletal muscle. Muscles were minced and digested in a mixture of type I collagenase and dispase B (Roche Applied Science). Cells were then filtered from debris, centrifuged and cultured F-10 Ham’s medium (Thermo Scientific), supplemented with 20% fetal bovine serum, 4 ng/ml basic fibroblast growth factor and 1% penicillin-streptomycin (Thermo Scientific) on collagen-coated dishes.
AAV9-CB-mini-agrin and AAV9-Control virus were infected to 10-cm plates of aged myoblasts from 18–20 months old mice. After 6 h, fresh Ham’s complete medium was added to replace the virus solution and samples were collected after an additional 72 h.

Primary myoblasts were isolated from hind limb skeletal muscle of 6-week-old male mice as previously described (Yue et al., 2017 (link)). Muscle tissues were minced and digested in type II collagenase and Dispase B mixture (Roche). Digested cells were harvested and cultured in growth media, F-10 Ham’s medium (Thermo Fisher Scientific) supplemented with 20% fetal bovine serum (FBS, Atlanta), 4 ng/ml basic fibroblast growth factor (Thermo Fisher Scientific) and 1% penicillin-streptomycin (Thermo Fisher Scientific) on collagen-coated dishes. Primary myoblasts were isolated and purified after 2–3 times of pre-plate. For in vitro genetic deletion, 4-OHT (0.4 μM, Calbiochem) was added in culture medium for 1 day to induce Cre-mediated deletion. P53 (Pifithrin-α, Sigma) and ATM/ATR (CGK733, Sigma) inhibitor were used according to the manufactory’s protocol and treated the myoblast together with 4-OHT or Methanol. Muscle differentiation was induced using 80% confluence of isolated primary myoblasts in Dulbecco's Modified Eagle Medium (DMEM, Sigma,) supplemented with 2% horse serum (Sigma). Differentiated cells were kept in differentiation media for further analysis.