Primary myoblasts were routinely grown in Skeletal Muscle Cell Basal Medium (PromoCell) supplemented with 10% FBS (Gibco), 5% fetal calf serum (FCS), 50 μg/mL fetuin, 10 ng/mL epidermal growth factor (EGF), 1 ng/mL basic fibroblast growth factor (bFGF), 10 μg/mL insulin, 400 ng/mL dexamethasone, 2 mM glutamine, 100 U/mL penicillin G, and 100 μg/mL streptomycin and used up to the seventh passage. To induce skeletal muscle differentiation, primary myoblasts at 70% confluence were washed three times in PBS and shifted to freshly prepared DMEM 4.5 g/L glucose supplemented with 5% horse serum (Gibco), 10 μg/mL insulin, 100 U/mL penicillin G, and 100 μg/mL streptomycin. After 2 to 4 days in these culture conditions, the appearance of multinucleated myotubes was noted. Because these myotubes, unless innervated, were short lived in culture, the AMO treatment was begun simultaneously with the differentiation process.
Skeletal muscle cell basal medium
Manufactured by PromoCell
Sourced in Austria
Skeletal Muscle Cell Basal Medium is a specialized cell culture medium designed to support the growth and maintenance of skeletal muscle cells in vitro. It provides the essential nutrients, growth factors, and other components required for the optimal cultivation of these cells.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Glutamax, by Thermo Fisher Scientific (2 mentions)
Horse serum, by Thermo Fisher Scientific (1 mentions)
Bovine growth serum (bgs), by Thermo Fisher Scientific (1 mentions)
Pyruvate, by Merck Group (1 mentions)
Gentamicin, by Thermo Fisher Scientific (1 mentions)
Ham s f10 medium, by Thermo Fisher Scientific (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
4 protocols using skeletal muscle cell basal medium
1
Primary Myoblast Culture and Differentiation
2
Immortalized and Primary DM1 Myoblasts
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LHCN-M2 immortalized human satellite cells,72 (link) carrying 2 (CTG·CAG)5 alleles (5/5 in short), were grown on 0.1% (w/v) gelatin-coated plastic surfaces in skeletal muscle cell basal medium (PromoCell) with Supplement Mix (0.05 mL/mL fetal calf serum, 50 µg/mL fetuin (bovine), 10 ng/mL epidermal growth factor (recombinant human), 1 ng/mL basic fibroblast growth factor (recombinant human), 10 µg/mL insulin (recombinant human), 0.4 µg/mL dexamethasone), supplemented with 1% (v/v) GlutaMAX and 15% (v/v) bovine growth serum (Thermo Scientific) at 7.5% CO2 and 37°C.
Primary DM1 myoblasts (13/800),73 (link) were grown on 0.1% (w/v) gelatin-coated plastic surfaces in Ham's F10 medium (Gibco) supplemented with GlutaMAX and 20% (v/v) bovine growth serum (Thermo Scientific) at 7.5% CO2 and 37°C.
Immortalized DM500 mouse myoblasts expressing a human DM1 genomic fragment carrying a (CTG·CAG)n repeat of approximately 500 triplets74 (link) were grown on 0.1% (w/v) gelatin-coated plastic surfaces in proliferation medium containing DMEM (Gibco) supplemented with 20% (v/v) fetal bovine serum (PAA, Pasching, Austria), 4 mML -glutamine (Gibco), 1 mM pyruvate (Sigma), 50 µg/mL gentamicin (Gibco), 20 units/mL γ-interferon (BD Biosciences) and 2% (v/v) chicken embryo extract (Sera Laboratories International) at 7.5% CO2 and 33°C.
Primary DM1 myoblasts (13/800),73 (link) were grown on 0.1% (w/v) gelatin-coated plastic surfaces in Ham's F10 medium (Gibco) supplemented with GlutaMAX and 20% (v/v) bovine growth serum (Thermo Scientific) at 7.5% CO2 and 37°C.
Immortalized DM500 mouse myoblasts expressing a human DM1 genomic fragment carrying a (CTG·CAG)n repeat of approximately 500 triplets74 (link) were grown on 0.1% (w/v) gelatin-coated plastic surfaces in proliferation medium containing DMEM (Gibco) supplemented with 20% (v/v) fetal bovine serum (PAA, Pasching, Austria), 4 mM
3
Myogenic Induction in Skeletal Muscle Cells
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hSkMC lines at 70% of confluence were cultured with a specific myogenic medium (MM): Skeletal Muscle Cell Basal Medium (PromoCell GmbH, cod. C 23260) supplemented with 10 µg/ml insulin, 100 IU/ml penicillin and 100 μg/ml streptomycin. The medium was refreshed twice a week. The expression of the myogenic phenotype was evaluated by microscopic observations of the multinucleated cells formation, by immunofluorescence of the myosin heavy chain (MHC) and by gene expression analysis, after 10 days of induction (Table 1 ).
4
Isolation of Human Primary Myoblasts
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Human primary myoblasts were kindly provided by Dr. Karim Bouzakri (University of Geneva), isolated as previously described 22 (link) from a skeletal muscle biopsy of non-diabetic patients undergoing surgery after informed consent by Ethics Committee. Briefly, after explant, the human skeletal muscle biopsy was maintained in 20 mL of physiological solution at +4°C for 2 h to allow blood coagulation. It was then processed under sterile hood with a scalpel, to isolate single muscle fibers from connective, adipose, and blood vessel tissues. The isolated fibers were maintained in a solution 0.05% Trypsin-EDTA at 37°C for 3 h to extract single-cell myoblast precursors. The surnatant cell suspension was then transferred into a new tube, added of an equal amount of fetal calf serum (FCS, Life Technologies), and centrifuged at 200 g for 5 min. The pellet was resuspended in 10 mL of skeletal muscle cell basal medium (PromoCell), and pre-plated in a bacterial petri dish for 2 h, the time required to have fibroblast adhesion and therefore exclusion.
Afterwards, medium with myoblasts still in suspension was collected and myoblasts plated in a 25mm 2 tissue culture flask. Human primary myoblasts were cultured for at least 7 days, with medium changes every 2-3 days. Cells were split before confluence by Trypsin-EDTA 0.05%.
Afterwards, medium with myoblasts still in suspension was collected and myoblasts plated in a 25mm 2 tissue culture flask. Human primary myoblasts were cultured for at least 7 days, with medium changes every 2-3 days. Cells were split before confluence by Trypsin-EDTA 0.05%.
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