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The C2C12 cell line, an immortalized mouse myoblast cell line derived from the thigh muscle of a 2-month-old mouse of the C3H mouse strain, is extensively utilized in biomedical research for its unique cell differentiation properties. C2C12 myoblast cells proliferate rapidly and exhibit typical myoblast characteristics under high serum conditions. Upon shifting to low serum conditions or starvation, C2C12 cells initiate myogenic differentiation, transitioning into myotubes, which are precursors to contractile skeletal muscle cells.
C2C12 cells incorporate exogenous cDNA and nucleic acids through transfection easily, making them a good choice for gene expression studies and investigations into myoblasts and myotubes differentiation. The differentiation process is marked by the expression of myogenic markers such as Myf5, MyoD, Myogenin, and Mrf4, alongside muscle-specific markers like Csrp3 and Mef2a, which are essential in studying different muscle phenotypes and skeletal muscle regeneration.
The unique shape of C2C12 myoblasts and their transformation into myoblast cell rings and subsequently into mature myotubes in serum-supplemented media underscore the dynamic nature of these cells and their potential in skeletal muscle research.
Researchers use substrates like gelatin hydrogels for C2C12 cell cultures to simulate in vivo muscle conditions, enabling detailed studies of muscle cell development and extracellular matrix effects. Metabolic profiling reveals key insights into the pathways involved in muscle formation and recovery, focusing on essential proteins and calcium's role in contraction. Gene silencing techniques further illuminate the differentiation process, highlighting the significance of SMAD1 phosphorylation in muscle regeneration, crucial for understanding recovery in muscle wasting and injury.
In summary, the C2C12 cell line serves as a critical tool in the realm of biomedical research, offering a versatile platform for exploring the intricacies of muscle formation, differentiation, gene expression, and the profound impact of various factors on the skeletal muscle cell lineage, including the pivotal role of myofilaments, intermediate filament proteins, and the overall organismal context in which these cellular processes unfold.
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- cultureMedium: RPMI 1640, w: 2.0 mM stable Glutamine, w: 2.0 g/L NaHCO3 (Cytion article number 820700a)
- supplements: Supplement the medium with 10% FBS
- dissociationReagent: Accutase
- doublingTime: 24 hours
- subculturing: Remove the old medium from the adherent cells and wash them with PBS that lacks calcium and magnesium. For T25 flasks, use 3-5 ml of PBS, and for T75 flasks, use 5-10 ml. Then, cover the cells completely with Accutase, using 1-2 ml for T25 flasks and 2.5 ml for T75 flasks. Let the cells incubate at room temperature for 8-10 minutes to detach them. After incubation, gently mix the cells with 10 ml of medium to resuspend them, then centrifuge at 300xg for 3 minutes. Discard the supernatant, resuspend the cells in fresh medium, and transfer them into new flasks that already contain fresh medium.
- seedingDensity: 1 x 104 cells/cm2 will yield in a confluent layer in about 4 days
- fluidRenewal: Every 3 to 5 days
- postThawRecovery: After thawing, plate the cells at 5 x 104 cells/cm2 and allow the cells to recover from the freezing process and to adhere for at least 24 hours.
- freezeMedium: As a cryopreservation medium, use complete growth medium (including FBS) + 10% DMSO for adequate post-thaw viability, or CM-1 (Cytion catalog number 800100), which includes optimized osmoprotectants and metabolic stabilizers to enhance recovery and reduce cryo-induced stress.
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