Mouse c2c12 myoblast

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Mouse C2C12 myoblasts are a well-established cell line derived from mouse skeletal muscle. They are commonly used as a model system for the study of myogenesis and muscle cell differentiation.

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C2C12 myoblasts (138 citations) C2C12 mouse myoblast cells (28 citations) C2C12 mouse myoblast cell line (26 citations) C2C12 mouse skeletal myoblasts (8 citations)

86 protocols using mouse c2c12 myoblast

Differentiation of C2C12 and 3T3-L1 Cells

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Mouse C2C12 myoblasts and 3T3-L1 preadipocytes were obtained from ATCC (Manassas, VA, USA). C2C12 cells were cultured in DMEM supplied with 10% FBS and 1% P/S. After 70‒80% confluence, the C2C12 cells were incubated with DMEM containing 1% P/S and 2% heat-inactivated HS for 4 days, to induce differentiation. Medium was changed every other day. The fully differentiated cells were treated with the different concentration of compounds for 24 h.
3T3-L1 preadipocytes were cultured in DMEM supplied with 10% CS and 1% P/S.2 Days post confluence, the cells were incubated with DMEM supplied with 10% FBS and DMI (1 μM dexamethasone, 0.5 mM 3-isobutyl-1-methylxanthine, and 5 μg/mL insulin) for 2 days. Subsequently, cells were maintained in DMEM supplied with 10% FBS and 5 μg/mL insulin for 6 days, and medium was refreshed every other day. On day 8, fully differentiated adipocytes were treated with compounds at indicated concentrations for 24 h.

Fang Z.J., Shen S.N., Wang J.M., Wu Y.J., Zhou C.X., Mo J.X., Lin L.G, & Gan L.S. (2019). Triterpenoids from Cyclocarya paliurus that Enhance Glucose Uptake in 3T3-L1 Adipocytes. Molecules, 24(1), 187.

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C2C12 Myoblast Differentiation Protocol

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Mouse C2C12 myoblasts (ATCC, Manassas, VA, USA) were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 50 U/ml penicillin, and 50 µg/ml streptomycin. When cells reached at confluence, the medium was switched to the differentiation medium containing DMEM and 2% horse serum, which was changed every other day.

Li Y., Park J., Wu Y., Cui J., Jia N., Xi M, & Wen A. (2017). Identification of AMPK activator from twelve pure compounds isolated from Aralia Taibaiensis: implication in antihyperglycemic and hypolipidemic activities. The Korean Journal of Physiology & Pharmacology : Official Journal of the Korean Physiological Society and the Korean Society of Pharmacology, 21(3), 279-286.

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Cell Culture Protocols for Cell Lines

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Human embryonic kidney cells (HEK293T), mouse embryonic fibroblasts (NIH‐3T3) and mouse preadipocytes (3T3‐L1) were purchased from China Center for Type Culture Collection and maintained at 37°C and 5% CO₂ in Dulbecco's modified Eagle's medium (DMEM) containing antibiotics (penicillin and streptomycin sulfate), l‐glutamine, sodium pyruvate and 10% foetal bovine serum (FBS). Mouse C2C12 myoblasts were purchased from ATCC and maintained at 37°C and 5% CO₂ in DMEM containing antibiotics (penicillin and streptomycin sulphate), l‐glutamine, sodium pyruvate and 20% FBS.

Zhu H., Liu D., Sui M., Zhou M., Wang B., Qi Q., Wang T., Zhang G., Wan F, & Zhang B. (2023). CRISPRa‐based activation of Fgf21 and Fndc5 ameliorates obesity by promoting adipocytes browning. Clinical and Translational Medicine, 13(7), e1326.

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Culturing and Differentiating Mouse C2C12 Myoblasts

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Mouse C2C12 myoblasts were purchased from the ATCC (Manassas, VA, USA) and cultured in DMEM supplemented with 100 U/ml penicillin and 100 μg/ml streptomycin and 10% FBS. Cell cultures were maintained at 37°C in a humidified atmosphere of 95% air and 5% CO₂. Medium was changed every 2 days. For the study of muscle cells, cells were differentiated as previous report.^{6 (link)}

Wang X., Li H., Zheng A., Yang L., Liu J., Chen C., Tang Y., Zou X., Li Y., Long J., Liu J., Zhang Y, & Feng Z. (2014). Mitochondrial dysfunction-associated OPA1 cleavage contributes to muscle degeneration: preventative effect of hydroxytyrosol acetate. Cell Death & Disease, 5(11), e1521-.

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Myoblast-Colon Cancer Cell Coculture

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Mouse C2C12 myoblasts and CT26 colon carcinoma cells were obtained from ATCC (Manassas, VA, USA). To build the coculture model, myoblasts were seeded (20,000 cells/cm 2 ) in the plate and CT26 cells were seeded (20,000 cells/cm 2 ) in transwells (Corning, #3450) on another plate. After 24 h of independent culture, the transwells were placed into the plates containing myoblasts and fresh medium with or without calpain inhibitor (CAST, Millipore, 208902, 1 μM; calpeptin (CAPT), Sigma-Aldrich, C8999, 50 μM). After 24 h of coculture, the myoblasts were stained with JC-1 or harvested for further analysis.
Our study consists of five groups: NC group, sham myoblasts (empty transwell and no calpain inhibitor); CO group, myoblasts cocultured with CT26 cells (no calpain inhibitor); CAST group, myoblasts cocultured with CT26 cells (with CAST); CAPT group, myoblasts cocultured with CT26 cells (with CAPT), and CC group, myoblasts cocultured with CT26 cells (with CAST and CAPT).

Zeng X., Zhao L., Chen Z., Kong L, & Chen S. (2022). Calpain inhibitors inhibit mitochondrial calpain activity to ameliorate apoptosis of cocultured myoblast. The Chinese journal of physiology, 65(5).

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Estrogen Regulation of Myoblast Differentiation

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Mouse C2C12 myoblasts (ATCC, Manassas, VA, USA) were maintained in high-glucose Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum and penicillin/streptomycin (growth medium) at 37°C in 5% CO 2 . When the cells reached confluence, the medium was switched to high-glucose DMEM supplemented with 2% horse serum and penicillin/streptomycin (differentiation medium) to induce C2C12 myotubes for 7 days and replaced with the fresh differentiation medium every 2 days. Infection of differentiated myoblastic C2C12 cells with Ad-caERα or fluorescent protein DsRed (Ad-DsRed) at m.o.i.30 (n = 3 for each group) was performed as described previously [6, 7] . Then, cells were treated with 1 μM ICI 182,780 (ICI) (Nacalai tesque, Kyoto, Japan) or vehicle for 48 h. Prior to estrogen treatment (10 nM 17β-estradiol (E2) (Sigma-Aldrich, St. Louis, MO, USA)), cells were maintained in phenol red-free DMEM containing 2% dextrancoated charcoal-stripped serum for 48 h and treated with combination of 1 μM ICI and 10 μg/mL puromycin (Sigma-Aldrich, St. Louis, MO, USA) for 4 h (n = 3 for each group) [13] .

Nagai S., Ikeda K., Horie-Inoue K., Takeda S, & Inoue S. (2018). Estrogen signaling increases nuclear receptor subfamily 4 group A member 1 expression and energy production in skeletal muscle cells. Endocrine journal, 65(12).

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Differentiating Mouse C2C12 Myoblasts

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Mouse C2C12 myoblasts (American Type Culture Collection, Manassas, VA, USA) were grown and passaged in Dulbecco’s modified Eagle medium (DMEM) containing 4.5 g/L glucose and supplemented with 10% fetal bovine serum (FBS; Atlanta Biologicals, Lawrenceville, GA, USA) plus antibiotics (100 U/ml penicillin, 100 mg/ml streptomycin; invitrogen, Carlsbad, CA, USA). At 95% confluence, cells were induced to differentiate into myotubes by replacing the growth media with DMEM containing 4.5 g/L glucose and supplemented with 2% horse serum (Invitrogen) and antibiotics for 3 days before treatments with the media changed daily. Cells were differentiated for at least 4 days to form mature myotubes [32 (link)].

Xie Y., Perry B.D., Espinoza D., Zhang P, & Price S.R. (2018). Glucocorticoid-induced CREB activation and myostatin expression in C2C12 myotubes involves phosphodiesterase-3/4 signaling. Biochemical and biophysical research communications, 503(3), 1409-1414.

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Differentiating Mouse C2C12 Myoblasts

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Differentiation and Treatment of C2C12 Myotubes

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Mouse C2C12 myoblasts (American Type Culture Collection) were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FBS. C2C12 myotube was obtained by culturing myoblasts in DMEM containing 2% horse serum for at least 5 days with daily media change. On the fifth day, differentiation of cells could be confirmed by multinucleated contracted myotubes. C2C12 myotubes were then treated with selected peptide candidates at indicated concentrations for 30 minutes. As a positive control, 2 μg/ml recombinant human globular adiponectin and indicated concentrations of ADP355 were used.

Kim S., Lee Y., Kim J.W., Son Y.J., Ma M.J., Um J.H., Kim N.D., Min S.H., Kim D.I, & Kim B.B. (2018). Discovery of a novel potent peptide agonist to adiponectin receptor 1. PLoS ONE, 13(6), e0199256.

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C2C12 Myoblast Differentiation and Psmg1 Knockdown

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Studies were performed using mouse C2C12 myoblasts (RRID: CVCL_0188) (American Type Culture Collection, Manassas, VA, USA). C2C12 mouse myoblasts were cultured in Dulbecco's modified Eagle's medium (Invitrogen) with 10% fetal bovine serum (Invitrogen), 2 mmol/L L-glutamine (WaKo Pure Chemical Industries, Ltd), 100 units/mL penicillin, and 0.1 mg/mL streptomycin (WaKo Pure Chemical Industries, Ltd). The cells were cultured at 37 °C in a 5% CO₂ atmosphere. C2C12 myoblasts were grown in 12-well plates with Dulbecco's modified Eagle's medium and supplemented with 2% horse serum for 4 days, inducing differentiation. C2C12 myotubes were transfected for 24 hours with 15 pmol of Psmg1 siRNA (MSS225905, MSS225906, MSS225907; Invitrogen) in each well or a negative control siRNA (Invitrogen) using Lipofectamine RNAiMAX (Invitrogen) according to the manufacturer's instructions.

Li Q., Ishii K.A., Kamoshita K., Takahashi K., Abuduwaili H., Takayama H., Galicia-Medina C.M., Tanida R., Ko Oo H., Gafiyatullina G., Yao X., Abuduyimiti T., Hamazaki J., Goto H., Nakano Y., Takeshita Y., Harada K., Murata S, & Takamura T. (2023). PAC1 Deficiency Protects Obese Male Mice From Immobilization-Induced Muscle Atrophy by Suppressing FoxO–Atrogene Axis. Endocrinology, 164(6), bqad065.

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