Dispase b

Primary myoblasts were isolated from hind limb skeletal muscle of male Linc-RAM KO and WT littermates at 3 weeks old, minced, and digested in a mixture of type I collagenase and Dispase B (Roche Applied Science). Cells were filtered from debris, centrifuged, and cultured in growth medium (F-10 Ham's medium supplemented with 20% fetal bovine serum, 4 ng ml⁻¹ basic fibroblast growth factor and 1% penicillin–streptomycin) on collagen-coated cell culture plates at 37 °C in 5% CO_2.

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.

Hindlimb skeletal muscles were minced and digested with a mixture of type I collagenase and dispase B (Roche Applied Science, Basel, Switzerland). The obtained cells were filtered, centrifuged, and cultured in growth medium (F-10 Ham's medium supplemented with 20% FBS, 4 ng/ml basic fibroblast growth factor, and 1% penicillin–streptomycin) on collagen-coated cell culture plates at 37 °C, 5% CO₂. The cell differentiation was induced in differentiation medium (DM) containing 2% HS and then cultured for 36 and 72 h, respectively.