Skgm 2 skeletal muscle cell growth medium 2 bulletkit

Human skeletal muscle myoblasts (HSMMs) were placed in a 6-well plate at a concentration of 6,250 cells/cm² and were incubated in the SkGM™-2 Skeletal Muscle Cell Growth Medium-2 BulletKit™ supplemented with 10% fetal bovine serum (Lonza, Cat. CC-3245) at 37 °C in a 95% humidified atmosphere of 5% CO₂. Cell viability of HSMMs was determined by the MTT assay (Sigma, Cat. TOX1-1 KT). In brief, HSMMs were plated at a density of 30,000 cells/cm², respectively, in 96-well plates and incubated for 24 h. FSME (0–5,000 ng/ml) and ASME (0–5,000 ng/ml) were added for 24–72 h. After incubation, cells were treated with 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT, 100 μL, 0.5 mg/ml) for 4 h at 37°C. The produced dark blue formazan crystals were solubilized by 100 μL DMSO. The absorbance at 570 nm was measured with a microplate reader. Cells without FSME or ASME treatment served as controls.

HEK293T (ATCC CRL-3216™) and HEK293T-derived CLCN5 GFP reporter cells, described recently (42 (link)), were cultured in Dulbecco’s modified Eagle’s medium with 10% fetal bovine serum (FBS), 2 mM l-glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin (Thermo Fisher Scientific) at 37°C in an incubator with 5% CO₂. Human IMR90 cells (lung fibroblasts; ATCC CCL-186) were cultured in Eagle’s minimum essential medium supplemented with 10% FBS, 2 mM l-glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin. Human CD34⁺ progenitor cells from mobilized peripheral blood (Lonza, catalog # 4Y-101C) were cultured in serum-free medium (Stem Cell Technology, catalog # 09605) supplemented with 1× StemSpan™ CD34⁺ Expansion Supplement (Stem Cell Technology, catalog # 02691). Human skeletal muscle myoblasts (Lonza, catalog # CC-2580) were cultured with an SkGM™-2 Skeletal Muscle Cell Growth Medium-2 BulletKit™ (Lonza, catalog # CC-3245).