C2c12 mouse myoblast cells

Manufactured by American Type Culture Collection

Sourced in United States

C2C12 mouse myoblast cells are a well-established in vitro model for the study of skeletal muscle development and differentiation. These cells are derived from the skeletal muscle of a C3H mouse and can be induced to differentiate into mature myotubes under appropriate culture conditions.

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28 protocols using c2c12 mouse myoblast cells

Culturing Trypanosoma cruzi in C2C12 Cells

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Tc SylvioX10 isolate was purchased from ATCC (Manassas, VA). Mouse myoblast C2C12 cells (ATCC CRL-1772) were seeded in T75 flasks containing 10 mL of RPMI 1640 with L-glutamine supplemented with 10% fetal bovine serum (FBS), 1% penicillin-streptomycin, and 1% sodium pyruvate (all media components from ThermoFisher Scientific, Waltham, MA), and cultured at 37 °C, 5% CO₂. At 70% confluence, cells were infected with Tc (1:3, cell: parasite ratio). Up to 50% of the medium was changed on alternate days. Tc replicates as an intracellular amastigote and differentiates from infective trypomastigotes that are released in the medium by cell lysis. Medium-containing trypomastigotes were collected in 15 mL conical tubes and sequentially centrifuged at room temperature at 290 rcf (g force) for 2 min to remove cell debris and at 3220 rcf for 8 min to pellet the parasites. Parasites were resuspended in 500 μL of PBS, and viable, parasites (moving, thin, free flagellum) were counted using a hemocytometer.

Rios L.E., Lokugamage N., Choudhuri S., Chowdhury I.H, & Garg N.J. (2023). Subunit nanovaccine elicited T cell functional activation controls Trypanosoma cruzi mediated maternal and placental tissue damage and improves pregnancy outcomes in mice. NPJ Vaccines, 8, 188.

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HEK293T and C2C12 cell culture

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Human embryonic kidney 293T (HEK293T) cells, mouse myoblast C2C12 cells (ATCC, Manassas, VA) and their derivatives were cultured under an atmosphere of 5% CO 2 at 37°C in Dulbecco's modified Eagle's medium (DMEM) (Sigma-Aldrich, St.
Louis, MO) supplemented with 10% (v/v) fetal bovine serum (Invitrogen, Carlsbad, CA), 55 µM β-mercaptoethanol (GIBCO, Grand Island, NY), 2 mM L-glutamine, 0.1 mM MEM non-essential amino acid, penicillin (10 U ml -1 ) and streptomycin (0.1 mg ml -1 ) (Sigma). Cells stably overexpressing 3×Flag-RNF207 were established by a retroviral expression system using pMX-puro with puromycin (8 µg ml -1 , Sigma) selection as described previously [26] .

Mizushima W., Takahashi H., Watanabe M., Kinugawa S., Matsushima S., Takada S., Yokota T., Furihata T., Matsumoto J., Tsuda M., Chiba I., Nagashima S., Yanagi S., Matsumoto M., Nakayama K.I., Tsutsui H, & Hatakeyama S. (2016). The novel heart-specific RING finger protein 207 is involved in energy metabolism in cardiomyocytes. Journal of molecular and cellular cardiology, 100.

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Transfection of C2C12 Myoblasts

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Mouse myoblast C2C12 cells (American Type Culture Collection, LGC Standards, Teddington, UK) were maintained in Dulbecco's modified Eagle's medium in 10% fetal bovine serum with 50 units/ml penicillin and 50 μl/ml streptomycin (GIBCO‐BRL, Grand Island, NY) at 37°C in a 5% CO₂‐humidified atmosphere. Cells were transfected using Lipofectamine 2000 transfection reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's recommendations.

Park J.E., Park J.S., Jang S.Y., Park S.H., Kim J., Ki C, & Kim D. (2019). A novel SMAD6 variant in a patient with severely calcified bicuspid aortic valve and thoracic aortic aneurysm. Molecular Genetics & Genomic Medicine, 7(5), e620.

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Energy Drink Effects on Myoblast Differentiation

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The mouse myoblast C2C12 cells (American Type Culture Collection; Manassas, VA, USA) were cultured in high glucose Dulbecco’s modified Eagle growth medium (DMEM; Gibco, USA) supplemented with 20% fetal bovine serum (FBS; Gibco, USA) and 1% penicillin–streptomycin (Gibco, USA). To induce differentiation, cells were switched to differentiation media which consists of high glucose DMEM (Gibco, USA) supplemented with 2% horse serum (Thermo Fisher Scientific, USA) and 1% penicillin–streptomycin (Gibco, USA) for 4 days. The differentiation media was changed every two days.
For energy drink treatments, cells were seeded at a density of 100,000 cells/well in a 24-well plate and incubated at 37 °C under 5% CO₂ for 24 h in growth media. Growth medium was then replaced with differentiation medium containing respective energy drinks. Cells were treated with either RedBull, RedBull Zero, Monster Energy, Monster Ultra Paradise, Rockstar, Rockstar Sugar Free, Celsius Live Fit, or Celsius Heat at 1:50 and 1:5 dilutions. Cells were differentiated for 4 days.

Park S.Y., Karantenislis G., Rosen H.T, & Sun H. (2023). Effects of energy drinks on myogenic differentiation of murine C2C12 myoblasts. Scientific Reports, 13, 8481.

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Cell Culture Conditions for RMS and Control Cells

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FN-RMS cell lines (RD, RMS-YM, and Rh18), FP-RMS cell lines (Rh30 and RM2), Mouse myoblast C2C12 cells, and human embryonic kidney HEK293 cells were cultured in high-glucose Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FBS, penicillin (100 U/ml), and streptomycin (10 mg/ml) at 37 °C in a humidified atmosphere containing 5% CO₂. RD, Rh-18, Rh30 and RM2 cell lines were kind gifts from Dr. Peter Houghton (The Research Institute at Nationwide Children’s Hospital, Columbus, OH). The RMS-YM and HEK293 cell lines were obtained from RIKEN BioResource Center (Tsukuba, Japan). Mouse myoblast C2C12 cells and human embryonic kidney HEK293 were purchased from the American Type Culture Collection (Manassas, VA).

Ouchi K., Miyachi M., Yagyu S., Kikuchi K., Kuwahara Y., Tsuchiya K., Iehara T, & Hosoi H. (2020). Oncogenic role of HMGA2 in fusion-negative rhabdomyosarcoma cells. Cancer Cell International, 20, 192.

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Cytotoxicity Evaluation of Liposomes

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C2C12 mouse myoblast cells (ATCC, Manassas, VA, USA) were cultured in DMEM (20% FBS and 1% Penicillin Streptomycin)(Invitrogen, Carlsbad, CA, USA). Cells were seeded in a 24-well plate at 5 × 10⁴ cells/mL in 800 mL media. The cells were then cultured in DMEM (2% horse serum and 1% Penicillin Streptomycin) for 10–14 days for myotubule differentiation.
PC12 rat adrenal gland pheochromocytoma cells (ATCC, Manassas, VA, USA) were cultured in DMEM (12.5% horse serum, 2.5% FBS and 1% Penicillin Streptomycin) and seeded in a 24 well-plate at 2 × 10⁴ cells/mL in 800 mL media. The cells were then cultured in DMEM (1% horse serum, 1% Penicillin Streptomycin, and 50 ng/mL nerve growth factor) for 7 days.
Cytotoxicity measurements were performed by exposing 100 μL of liposomes to C2C12 or PC12 cells (800 μL of their respective cell culture media) by a 24-well Transwell® membrane (Costar 3495, pore size 0.4 μm). Irradiation was performed with 730 nm light at 50 mW/cm² for 5 min. Cell viability was measured by the MTS assay after 96 h incubation.

Rwei A.Y., Wang B., Ji T., Zhan C, & Kohane D.S. (2017). Enhanced triggering of local anesthetic particles by photosensitization and photothermal effect using a common wavelength. Nano letters, 17(11), 7138-7145.

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C2C12 Myoblast Culture and Inhibitor Treatment

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C2C12 mouse myoblast cells (ATCC, UK) were cultured at 37°C under a humidified atmosphere of 5% CO₂ in Dulbecco's modified Eagles's medium (DMEM/4.5 g/l glucose + GlutaMAX, Gibco, UK) supplemented with 10% fetal bovine serum, 100 units/ml penicillin and 100 mg/ml streptomycin. Cells (100,000) were plated 24 h prior to experiment in 6-well plates with 2 ml medium. For experiments inhibitors were added at the following concentrations: 1 μM rotenone, 1 mM theonoyltrifluoroacetone (TTFA), 5 μM FCCP, 5 μM Antimycin A. Cells were then incubated for 5 min at 37°C, washed with PBS and scraped in 200 μl fresh PBS on ice prior to CoQ extraction.

Burger N., Logan A., Prime T.A., Mottahedin A., Caldwell S.T., Krieg T., Hartley R.C., James A.M, & Murphy M.P. (2020). A sensitive mass spectrometric assay for mitochondrial CoQ pool redox state in vivo. Free Radical Biology & Medicine, 147, 37-47.

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Silencing of SDC4 in C2C12 Mouse Myoblasts

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C2C12 mouse myoblast cells (ATCC, Manassas, VA, USA) were cultured in Dulbecco's modified Eagle's medium (4.5 g·L⁻¹ glucose with glutamine; Lonza, Basel, Switzerland) containing 50 μg·mL⁻¹ gentamycin (Lonza) and supplemented with 20% fetal bovine serum (Gibco, Thermo Fisher Scientific, Waltham, MA, USA). Differentiation was induced by shifting the confluent cultures to medium containing 2% horse serum (Sigma‐Aldrich). For SDC4 silencing the C2C12 cells were stably transfected with plasmids expressing short hairpin RNAs (shRNA) targeting mouse SDC4 (shSDC4#1 and shSDC#2), a scrambled target sequence, or the empty pRS vector using X‐tremeGENE transfection reagent (Roche, Basel, Switzerland). The transfected populations were selected in medium supplemented with 4 μg·mL⁻¹ puromycin (Sigma‐Aldrich). The plasmids were obtained from OriGene (TR513122; Rockville, MD, USA) and targeted the sequences 5′‐GAA CTG GAA GAG AAT GAG GTC ATT CCT AA‐3′ (shSDC4#1), 5′‐GCG GCG TGG TAG GCA TCC TCT TTG CCG TT‐3′ (shSDC4#2) and 5′‐GCA CTA CCA GAG CTA ACT CAG ATA GTA CT‐3′ (scrambled).

Keller‐Pinter A., Szabo K., Kocsis T., Deak F., Ocsovszki I., Zvara A., Puskas L., Szilak L, & Dux L. (2018). Syndecan‐4 influences mammalian myoblast proliferation by modulating myostatin signalling and G1/S transition. Febs Letters, 592(18), 3139-3151.

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C2C12 Mouse Myoblast Cell Culture

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The C2C12 mouse myoblast cells were purchased from ATCC (Manassas, VA, USA). The cell culture procedures were generally as described by Somwar et al. [16 (link)], with slight modifications [17 (link)]. The cells were grown in Delbecco’s modified Eagle Medium (DMEM, Thermo Fisher) supplemented with 10% FetalPlex (Gemini Bio Sciences, Sacramento, CA, USA) and 1% penicillin/streptomycin solution (Sigma). The cells were incubated at 37 ℃ at 5% CO₂ and passaged every 48–72 h.

Dedert C.J., Bagdady K.R, & Fisher J.S. (2023). Prior Treatment with AICAR Causes the Selective Phosphorylation of mTOR Substrates in C2C12 Cells. Current Issues in Molecular Biology, 45(10), 8040-8052.

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C2C12 and MEF Cell Treatment

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C2C12 mouse myoblast cells (ATCC, Virginia, U.S.A) and mouse embryonic fibroblasts (MEFs) were treated for 16 hours with vehicle control [0.5% DMSO (Sigma-Aldrich, Dorset, U.K.) pH 7] or 1 μM IOX3 (dissolved in 0.5% DMSO pH 7).

McMurray F., Demetriades M., Aik W., Merkestein M., Kramer H., Andrew D.S., Scudamore C.L., Hough T.A., Wells S., Ashcroft F.M., McDonough M.A., Schofield C.J, & Cox R.D. (2015). Pharmacological Inhibition of FTO. PLoS ONE, 10(4), e0121829.

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