Wt MEFs (ATCC® CRL-2991)

Organism: Mus musculus, mouse  /  Cell Type: fibroblast  /  Tissue: embryo/embryo fibroblast  / 

Permits and Restrictions

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Organism Mus musculus, mouse
Tissue embryo/embryo fibroblast
Cell Type fibroblast
Product Format frozen
Morphology fibroblast-like
Culture Properties adherent
Biosafety Level 2   [Cells contain SV40 viral DNA sequences]
Age embryo, 10.5-days gestation
Applications
This cell line can be used as a control to study the role of mitochondrial fusion in cell physiology.
Storage Conditions liquid nitrogen vapor phase
Images
Derivation
MEFs were derived from embryos 10.5 days gestation. Embryos were mechanically dispersed by repeated passage through a P1000 pipette tip and plated with MEF medium (DME, 10% FCS, 1x nonessential amino acid, 1 mM L-glutamine (Invitrogen/Gibco)). Cells were subsequently immortalized by transduction with SV-40 T antigen. 
Complete Growth Medium The base medium for this cell line is ATCC-formulated Dulbecco's Modified Eagle's Medium, Catalog No. 30-2002. To make the complete growth medium, add the following components to the base medium: bovine calf serum to a final concentration of 10%.
Subculturing

Volumes used in this protocol are for 75 cm2 flasks; proportionally reduce or increase amount of dissociation medium for culture vessels of other sizes.

  1. Remove and discard culture medium.
  2. Briefly rinse the cell layer with Ca++/Mg++ free Dulbecco's phosphate-buffered saline (D-PBS) or 0.25% (w/v) Trypsin - 0.53 mM EDTA solution to remove all traces of serum which contains trypsin inhibitor.
  3. Add 2.0 to 3.0 mL of Trypsin-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.
  6. Place culture vessels in incubators at 37°C.

Subcultivation ratio: A subcultivation ratio of 1:5 to 1:20 is recommended

Maintenance

The flask was seeded with cells (see specific batch information), grown, and completely filled with medium at ATCC to prevent loss of cells during shipping.

  1. Upon receipt, visually examine the culture for macroscopic evidence of any microbial contamination. Using an inverted microscope (preferably equipped with phase-contrast optics), carefully check for any evidence of microbial contamination.  Also, check to determine if the majority of cells are still attached to the bottom of the flask; during shipping the cultures are sometimes handled roughly and many of the cells often detach and become suspended in the culture medium (but are still viable).
  2. If the cells are still attached, aseptically remove all but 5 to 10 mL of the shipping medium. The shipping medium can be saved for reuse. Incubate the cells at 37°C in a 5% CO2 in air atmosphere until they are ready to be subcultured.
  3. If the cells are not attached, aseptically remove the entire contents of the flask and centrifuge at 125 x g for 5 to 10 minutes. Remove shipping medium and save.  Resuspend the pelleted cells in 10 mL of this medium and add to 25 cm2 flask.  Incubate at 37°C in a 5% CO2 in air atmosphere until cells are ready to be subcultured.
Cryopreservation
Freeze medium: complete growth medium, 90%; DMSO, 10%
liquid nitrogen vapor phase
Culture Conditions
Temperature: 37°C
Atmosphere: air, 95%; carbon dioxide (CO2), 5%
Name of Depositor D Chan
Passage History
­Embryos were mechanically dispersed by repeated passage through a P1000 pipette tip and plated with MEF medium (DME, 10% FCS, 1x nonessential amino acid, 1 mM L-glutamine (Invitrogen/Gibco)). Cells were subsequently immortalized by transduction with SV-40 T antigen. ­
Year of Origin 2000
References

Chen H, et al. Mitofusins Mfn1 and Mfn2 coordinately regulate mitochondrial fusion and are essential for embryonic development. J. Cell Biol. 160: 189-200, 2003. PubMed: 12527753

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

Chen H, et al. Mitofusins Mfn1 and Mfn2 coordinately regulate mitochondrial fusion and are essential for embryonic development. J. Cell Biol. 160: 189-200, 2003. PubMed: 12527753