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C2C12 clone 5 DYS shRNA (ATCC® CRL-3418)

Organism: Mus musculus, mouse  /  Tissue: skeletal muscle  / 

Permits and Restrictions

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Organism Mus musculus, mouse
Tissue skeletal muscle
Product Format frozen 1.0 mL
Morphology myoblast
Culture Properties adherent
Biosafety Level 1

Biosafety classification is based on U.S. Public Health Service Guidelines, it is the responsibility of the customer to ensure that their facilities comply with biosafety regulations for their own country.

Age adult
Strain C3H
Applications Functional studies of dysferlin and ferlin proteins. Cell line can be used to study membrane repair and muscle regeneration mechanisms.
Storage Conditions liquid nitrogen vapor phase
Images CRL-3418 Micrograph
Comments Modified genetically: Stably transfected with a dysferlin (DYSF) shRNA. ATCC CRL-3418 dysferlin shRNA Clone 5 cells show reduced dysferlin protein levels compared to the scrambled shRNA control cell line (ATCC CRL-3419).
Complete Growth Medium The base medium for this cell line is Dulbecco's Modified Eagle's Medium (DMEM; ATCC 30-2002). To make the complete growth medium for the first 3 to 5 days of culture, add the following components to the base medium: fetal bovine serum (FBS; ATCC 30-2020) to a final concentration of 10% and 0.5 µg/ml puromycin . After 3-5 days, use DMEM + 10% FBS with 2 µg/ml puromycin in order to maintain the presence of the DYS shRNA.
Subculturing
Volumes used in this protocol are for 75 cm2 flask; proportionally reduce or increase amount of dissociation medium for culture vessels of other sizes. Corning® T-75 flasks (catalog #430641) are recommended for subculturing this product.
  1. Remove and discard culture medium.
  2. Briefly rinse the cell layer with DPBS (ATCC catalog # 30-2200) to remove all traces of serum that contains trypsin inhibitor.
  3. Add 2.0 to 3.0 mL 0.25% (w/v) Trypsin- 0.53 mM EDTA solution to flask and observe cells under an inverted microscope until cell layer is dispersed (usually within 5 to 15 minutes).
    Note: To avoid clumping do not agitate the cells by hitting or shaking the flask while waiting for the cells to detach. Cells that are difficult to detach may be placed at 37°C to facilitate dispersal.
  4. Add 6.0 to 8.0 mL of complete growth medium and aspirate cells by gently pipetting.
  5. Add appropriate aliquots of the cell suspension to new culture vessels.
    Cultures can be established between 2.5 x 104 and 4.0 x 104 viable cells/cm2.
  6. Incubate cultures at 37°C.
Interval: Maintain cultures at a cell concentration between 2.0 x 104 and 3.8 x 105 cell/cm2.
Subcultivation Ratio: A subcultivation ratio of 1:3 to 1:8 is recommended
Medium Renewal: 2 to 3 times per week
Cryopreservation 95% complete medium + 5% DMSO
Culture Conditions
Atmosphere: air, 95%; carbon dioxide (CO2), 5%
Temperature: 37°C
Cells per Vial Approximately 2.0 x 106 cells
Volume 1.0 mL
Sterility Tests Bacteria and yeast: No growth
Mycoplasma: No growth
Viability ≥ 50%
Population Doubling Time 48-72 hours
Name of Depositor R Bashir, Department Biosciences, Durham University, Durham, UK
Year of Origin 2013
References

Defour A, et al. Dysferlin regulates cell membrane repair by facilitating injury-triggered acid sphingomyelinase secretion. Cell Death Dis 5: e1306, 2014. PubMed: 24967968

Notice: Necessary PermitsPermits

These permits may be required for shipping this product:

  • Customers located in the state of Hawaii will need to contact the Hawaii Department of Agriculture to determine if an Import Permit is required. A copy of the permit or documentation that a permit is not required must be sent to ATCC in advance of shipment.
Basic Documentation
Other Documentation
References

Defour A, et al. Dysferlin regulates cell membrane repair by facilitating injury-triggered acid sphingomyelinase secretion. Cell Death Dis 5: e1306, 2014. PubMed: 24967968