C2C12 Cells
Advancements in Muscle Research Through C2C12 Myotubes
Description | 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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Organism | Mouse |
Tissue | Muscle |
Applications | Transfection host |
Synonyms | C2c12, C2-C12, C12 |
Properties
Age | 2 months |
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Gender | Female |
Morphology | Myoblast-like |
Cell type | Myoblast |
Growth properties | Adherent |
Specification
Citation | C2C12 (Cytion catalog number 400476) |
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Biosafety level | 1 |
Genetic profile of the C2C12 mouse cell line
Culturing methods
Culture Medium | RPMI 1640, w: 2.1 mM stable Glutamine, w: 2.0 g/L NaHCO3 (Cytion article number 820700a) |
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Medium supplements | Supplement the medium with 10% FBS |
Passaging solution | Accutase |
Doubling time | 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. |
Split ratio | A split ratio of 1:3 to 1:5 is recommended |
Seeding density | 1 x 10^4 cells/cm^2 will yield in a confluent layer in about 4 days |
Fluid renewal | Every 3 to 5 days |
Freezing recovery | After thawing, plate the cells at 5 x 10^4 cells/cm^2 and allow the cells to recover from the freezing process and to adhere for at least 24 hours. |
Freeze medium | CM-1 (Cytion catalog number 800100) or CM-ACF (Cytion catalog number 806100) |
Handling of cryopreserved cultures |
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Quality control on C2C12 cells
Sterility | Mycoplasma contamination is excluded using both PCR-based assays and luminescence-based mycoplasma detection methods. To ensure there is no bacterial, fungal, or yeast contamination, cell cultures are subjected to daily visual inspections. |
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STR profile |
M_18-3: 16
M_4-2: 19. Mrz
M_6-7: 12
M_3-2: 14
M_19-2: 12
M_7-1: 26
M_1-1: 10
M_8-1: 17
M_2-1: 9
M_15-3: 25. Mrz
M_6-4: 18
M_11-2: 16
M_1-2: 16
M_17-2: 15
M_12-1: 16
M_5-5: 15
M_X-1: 25,26
M_13-1: 17
Human D4/D8: -
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Required products
Key Features of Freeze Medium CM-ACF
Serum-Free Formulation: Eliminates the variability and risks associated with serum, providing a defined and controlled environment for a wide array of cell types, including primary cells, stem cells, and established cell lines.
Broad Compatibility: Effective for a wide array of cell types, including primary cells, stem cells, and established cell lines.
High Viability: Optimized to maximize post-thaw cell recovery and viability, ensuring reliable experimental outcomes.
Ready-to-Use: Conveniently prepared and sterilized for immediate application, reducing preparation time and risk of contamination.
Enhanced Stability: Maintains consistent performance under standard cryopreservation conditions, ensuring reproducible results.
Long Shelf Life: CM-ACF is a serum-containing, ready-to-use cryopreservation medium that can be stored in the refrigerator for up to one year.
Using CM-ACF for Freezing Cells
To use CM-ACF for freezing both adherent and suspension cells, follow these steps:
Preparation:
For adherent cells: Wash and dissociate them from the culture substrate.
For suspension cells: Proceed directly to the next step.
Cell Counting: Ensure cells are at the proper concentration.
Centrifugation: Pellet the cells and resuspend them in CM-ACF freeze medium.
Cryovial Transfer: Transfer the resuspended cells into cryovials.
Freezing Process: Use a slow-freezing method before transferring the cells to long-term storage.
Freezing Methods
Method
Description
Steps
❄️ Manual Freezing
A step-by-step method involving gradual temperature reduction to ensure cell viability.
1. Place cells in freeze medium in a 4°C freezer for 40 minutes. 2. Transfer to a -80°C freezer for 24 hours. 3. Store cells in liquid nitrogen for long-term preservation.
🧊 Using Mr. Frosty
A convenient device that allows for controlled freezing rates without electrical power.
1. Prepare cells in cryovials with freeze medium. 2. Place cryovials in Mr. Frosty container. 3. Store at -80°C for 24 hours before transferring to liquid nitrogen.
🧬 Controlled-Rate Freezer
A high-precision freezer by Thermo Fisher or other manufacturers designed for controlled temperature reduction.
1. Program the device to gradually decrease the temperature. 2. Place prepared cells in the freezer. 3. After the freezing cycle, transfer cells to liquid nitrogen.
Long-Term Storage
Store the cryovials at temperatures below -130°C or in liquid nitrogen for long-term preservation.
Ingredients
Contains DMSO, Glucose, Salts
Buffering capacity: pH = 7.2 to 7.6
Cytion’s Freeze Medium CM-ACF offers a reliable solution for cryopreservation, ensuring high cell viability and functionality post-thaw for a wide range of research applications.
Initially designed to support the growth of human leukemic cells in both suspension and monolayer cultures, RPMI 1640 Medium has evolved through modifications by researchers and commercial suppliers to become suitable for a diverse range of mammalian cells. It is exceptionally compatible with cell lines such as HeLa, Jurkat, MCF-7, PC12, PBMC, astrocytes, and carcinomas.
RPMI 1640 Medium stands apart from other cell culture media due to its unique composition. It contains a substantial amount of phosphate, amino acids, and vitamins. Notably, it encompasses biotin, vitamin B12, and PABA, absent in Eagle's Minimal Essential Medium or Dulbecco's Modified Eagle Medium. Moreover, RPMI 1640 Medium exhibits significantly elevated concentrations of vitamins inositol and choline. However, it does not contain proteins, lipids, or growth factors. Consequently, supplementation with 10% Fetal Bovine Serum (FBS) is commonly required to provide optimal conditions for cell growth.
The buffering system of RPMI 1640 Medium relies on sodium bicarbonate (2.0 g/L) and necessitates a 5-10% CO2 environment to maintain a physiologically appropriate pH. The inclusion of the reducing agent glutathione further distinguishes this medium from others.
This RPMI 1640 medium contains 4.5 grams per liter of glucose.
Quality control
pH = 7.2 +/
- 0.02 at 20-25°C.
Each lot has been tested for sterility and absence of mycoplasma and bacteria.
Maintenance
Keep refrigerated at +2°C to +8°C in the dark. Freezing and warming up to +37° C minimize the quality of the product.
Do not heat the medium to more than 37° C or use uncontrollable sources of heat (e.g., microwave appliances).
If only a part of the medium is to be used, remove this amount from the bottle and warm it up at room temperature.
Shelf life for any medium except for the basic medium is 8 weeks from the date of manufacture.
Composition
Components
mg/L
Inorganic Salts
Calcium nitrate x 4H2O
100,00
Magnesium sulfate anhydrous
48,83
Potassium chloride
400,00
Sodium chloride
5450,00
di-Sodium hydrogen phosphate
800,49
Other Components
D(+)-Glucose anhydrous
4500,00
Glutathione (red.)
1,00
HEPES
2383,00
Phenol red
5,00
Sodium pyruvate
110,00
Amino Acids
L-Arginine x HCl
241,86
L-Asparagine x H2O
56,82
L-Aspartic acid
20,00
L-Cystine x 2HCl
65,19
L-Glutamine
300,00
L-Glutamic acid
20,00
Glycine
10,00
L-Histidine x HCl x H2O
20,27
L-Hydroxyproline
20,00
L-Isoleucine
50,00
L-Leucine
50,00
L-Lysine x HCl
40,00
L-Methionine
15,00
L-Phenylalanine
15,00
L-Proline
20,00
L-Serine
30,00
L-Threonine
20,00
L-Tryptophan
5,00
L-Tyrosine x 2Na
28,83
L-Valine
20,00
Vitamins
p-Aminobenzoic acid
1,00
D-(+)-Biotin
0,20
D-Calcium pantothenate
0,25
Choline chloride
3,00
Folic acid
1,00
myo-Inositol
35,00
Nicotinamide
1,00
Pyridoxine x HCl
1,00
Riboflavin
0,20
Thiamine x HCl
1,00
Vitamin B12
0,01
NaHCO3
1500,00
Initially designed to support the growth of human leukemic cells in both suspension and monolayer cultures, RPMI 1640 Medium has evolved through modifications by researchers and commercial suppliers to become suitable for a diverse range of mammalian cells. It is exceptionally compatible with cell lines such as HeLa, Jurkat, MCF-7, PC12, PBMC, astrocytes, and carcinomas.
RPMI 1640 Medium stands apart from other cell culture media due to its unique composition. It contains a substantial amount of phosphate, amino acids, and vitamins. Notably, it encompasses biotin, vitamin B12, and PABA, absent in Eagle's Minimal Essential Medium or Dulbecco's Modified Eagle Medium. Moreover, RPMI 1640 Medium exhibits significantly elevated concentrations of vitamins inositol and choline. However, it does not contain proteins, lipids, or growth factors. Consequently, supplementation with 10% Fetal Bovine Serum (FBS) is commonly required to provide optimal conditions for cell growth.
The buffering system of RPMI 1640 Medium relies on sodium bicarbonate (2.0 g/L) and necessitates a 5-10% CO2 environment to maintain a physiologically appropriate pH. The inclusion of the reducing agent glutathione further distinguishes this medium from others.
The unique composition of this RPMI formulation comprises 2.1 mM of stable Glutamine, 2.0 grams per liter of NaHCO3, and phenol red.
Quality control
pH = 7.2 +/
- 0.02 at 20-25°C.
Each lot has been tested for sterility and absence of mycoplasma and bacteria.
Maintenance
Keep refrigerated at +2°C to +8°C in the dark. Freezing and warming up to +37° C minimize the quality of the product.
Do not heat the medium to more than 37° C or use uncontrollable sources of heat (e.g., microwave appliances).
If only a part of the medium is to be used, remove this amount from the bottle and warm it up at room temperature.
Shelf life for any medium except for the basic medium is 8 weeks from the date of manufacture.
Composition
Components
mg/L
Inorganic Salts
Calcium nitrate x 4H2O
100,00
Magnesium sulfate anhydrous
48,83
Potassium chloride
400,00
Sodium chloride
5,950.00
di-Sodium hydrogen phosphate
800,49
Other Components
D(+)-Glucose anhydrous
2,000.00
Glutathione (red.)
1,00
Phenol red
5,00
NaHCO3
2,000.00
Amino Acids
L-Arginine x HCl
241,86
L-Asparagine x H2O
56,82
L-Aspartic acid
20,00
L-Cystine x 2HCl
65,19
L-Alanyl-L-Glutamine
447,00
L-Glutamic acid
20,00
Glycine
10,00
L-Histidine x HCl x H2O
20,27
L-Hydroxyproline
20,00
L-Isoleucine
50,00
L-Leucine
50,00
L-Lysine x HCl
40,00
L-Methionine
15,00
L-Phenylalanine
15,00
L-Proline
20,00
L-Serine
30,00
L-Threonine
20,00
L-Tryptophan
5,00
L-Tyrosine x 2Na
28,83
L-Valine
20,00
Vitamins
p-Aminobenzoic acid
1,00
D-(+)-Biotin
0,20
D-Calcium pantothenate
0,25
Choline chloride
3,00
Folic acid
1,00
myo-Inositol
35,00
Nicotinamide
1,00
Pyridoxine x HCl
1,00
Riboflavin
0,20
Thiamine x HCl
1,00
Vitamin B12
0.005
- A Gentle Alternative to Trypsin
Accutase is a cell detachment solution that is revolutionizing the cell culture industry. It is a mix of proteolytic and collagenolytic enzymes that mimics the action of trypsin and collagenase. Unlike trypsin, Accutase does not contain any mammalian or bacterial components and is much gentler on cells, making it an ideal solution for the routine detachment of cells from standard tissue culture plasticware and adhesion coated plasticware. In this blog post, we will explore the benefits and uses of Accutase and how it is changing the game in cell culture.
Advantages of Accutase
Accutase has several advantages over traditional trypsin solutions. Firstly, it can be used whenever gentle and efficient detachment of any adherent cell line is needed, making it a direct replacement for trypsin. Secondly, Accutase works extremely well on embryonic and neuronal stem cells, and it has been shown to maintain the viability of these cells after passaging. Thirdly, Accutase preserves most epitopes for subsequent flow cytometry analysis, making it ideal for cell surface marker analysis.
Additionally, Accutase does not need to be neutralized when passaging adherent cells. The addition of more media after the cells are split dilutes Accutase so it is no longer able to detach cells. This eliminates the need for an inactivation step and saves time for cell culture technicians. Finally, Accutase does not need to be aliquoted, and a bottle is stable in the refrigerator for 2 months.
Applications of Accutase
Accutase is a direct replacement for trypsin solution and can be used for the passaging of cell lines. Additionally, Accutase performs well when detaching cells for the analysis of many cell surface markers using flow cytometry and for cell sorting. Other downstream applications of Accutase treatment include analysis of cell surface markers, virus growth assay, cell proliferation, tumor cell migration assays, routine cell passage, production scale-up (bioreactor), and flow cytometry.
Composition of Accutase
Accutase contains no mammalian or bacterial components and is a natural enzyme mixture with proteolytic and collagenolytic enzyme activity. It is formulated at a much lower concentration than trypsin and collagenase, making it less toxic and gentler, but just as effective.
Efficiency of Accutase
Accutase has been shown to be efficient in detaching primary and stem cells and maintaining high cell viability compared to animal origin enzymes such as trypsin. 100% of cells are recovered after 10 minutes, and there is no harm in leaving cells in Accutase for up to 45 minutes, thanks to autodigestion of Accutase.
In summary
In conclusion, Accutase is a powerful solution that is changing the game in cell culture. With its gentle nature, efficiency, and versatility, Accutase is the ideal alternative to trypsin. If you are looking for a reliable and efficient solution for cell detachment, Accutase is the solution for you.
Phosphate-buffered saline (PBS) is a versatile buffer solution used in many biological and chemical applications, as well as tissue processing. Our PBS solution is formulated with high-quality ingredients to ensure a constant pH during experiments. The osmolarity and ion concentrations of our PBS solution are matched to those of the human body, making it isotonic and non-toxic to most cells.
Composition of our PBS Solution
Our PBS solution is a pH-adjusted blend of ultrapure-grade phosphate buffers and saline solutions. At a 1X working concentration, it contains 137 mM NaCl, 2.7 mM KCl, 8 mM Na2HPO4, and 2 mM KH2PO4. We have chosen this composition based on CSHL protocols and Molecular cloning by Sambrook, which are well-established standards in the research community.
Applications of our PBS Solution
Our PBS solution is ideal for a wide range of applications in biological research. Its isotonic and non-toxic properties make it perfect for substance dilution and cell container rinsing. Our PBS solution with EDTA can also be used to disengage attached and clumped cells. However, it is important to note that divalent metals such as zinc cannot be added to PBS as this may result in precipitation. In such cases, Good's buffers are recommended. Moreover, our PBS solution has been shown to be an acceptable alternative to viral transport medium for the transport and storage of RNA viruses, such as SARS-CoV-2.
Storage of our PBS Solution
Our PBS solution can be stored at room temperature, making it easy to use and access.
To sum up
In summary, our PBS solution is an essential component in many biological and chemical experiments. Its isotonic and non-toxic properties make it suitable for numerous applications, from cell culture to viral transport medium. By choosing our high-quality PBS solution, researchers can optimize their experiments and ensure accurate and reliable results.
Composition
Components
mg/L
Inorganic Salts
Potassium chloride
200,00
Potassium dihydrogen phosphate
200,00
Sodium chloride
8,000.00
di-Sodium hydrogen phosphate anhydrous
1,150.00