MEF (C57BL/6) [MEF-BL/6-1] (ATCC® SCRC-1008)

Organism: Mus musculus, mouse  /  Cell Type: Fibroblast  /  Tissue: Embryo  / 

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Organism Mus musculus, mouse
Tissue
Embryo
Cell Type Fibroblast
Product Format frozen
Morphology Fibroblast
Culture Properties Adherent
Biosafety Level 1
Age 14 days gestation embryo
Gender male and female mixed
Strain C57BL/6
Applications
The cells can be used as a feeder layer to support the growth of embryonic stem (ES) cells and for the maintenance of ES cells in the undifferentiated state. The growth of these cells must be arrested before they can be used as a feeder layer. ATCC has successfully treated the cells with mitomycin C for use as a feeder layer. If the MEFs are being used as a feeder layer for ES cells, it is not recommended to use them past passage no. 6 (P6).
Storage Conditions liquid nitrogen vapor phase
Derivation
The cell line was established by ATCC in 2003 from dissociated C57BL/6 mouse embryos.
Clinical Data
male and female mixed
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:
  • fetal bovine serum to a final concentration of 15%

  • This medium is formulated for use with a 5% CO2 in air atmosphere. (Standard DMEM formulations contain 3.7 g/L sodium bicarbonate and a 10% CO2 in air atmosphere is then recommended).
    Subculturing To insure the highest level of viability, be sure to warm media and Trypsin / EDTA to 37ºC before using it on the cells. Cells should be split when they reach confluency. A split base on seed density of 2 X 104 cells/cm2 is recommended.
    1. Remove and discard culture medium.
    2. Briefly rinse the cell layer with 1XPBS (SCRR-2201) solution to remove all traces of serum, which contain trypsin inhibitor.
    3. Add Trypsin-EDTA (0.25% Trypsin-0.53 mM EDTA solution, ATCC# 30-2101) solution to the flask (Table 1) and incubate for 2 minutes. Gently tapping the flask, observe cells under an inverted microscope. Cells usually detach in 2 to 3 minutes.
    4. Add an equal volume complete of the growth medium (Table 1) and rinse surface of the flask to detach all the cells. Gently pipetting up and down will break cell clumps. 
    5. Transfer all cells into a centrifuge bottle or tube and centrifuge at 270 x g for 5 minutes.
    6. Remove and discard the supernatant
    7. Add 10 mL complete growth medium to the cell pellet and with 10 mL pipette resuspend the cells gently (create a single-cell suspension).
    8. Add more complete growth medium (Table 1) to the cell suspension as needed to plate cells at approximately 0.8 X 104 cells/cm2.
    9. Place flasks in the incubator @ 37°C with a 5% CO2 in air atmosphere 

    Flask/Plate 

     

     

     

    Growth Area (cm2

     

     

     

     1xPBS (mL)

     

     

     

    Trypsin/EDTA (mL) 

     

     

     

    Equal vol. Complete Growth Medium (mL) 

     

     

     

    Growth Medium (mL) 

     

     

     

     T225

     

     

     

     225

     

     

     

     10 ± 0.2

     

     

     

     6 ± 0.2

     

     

     

     6 ± 0.2

     

     

     

     30

     

     

     

     75

     

     

     

     75

     

     

     

     5 ± 0.1

     

     

     

     3 ± 0.1

     

     

     

     3 ± 0. 1

     

     

     

     12

     

     

     

     T25

     

     

     

     25

     

     

     

     3 ± 0.1

     

     

     

     1.5 ± 0.1

     

     

     

     1.5 ± 0.1

     

     

     

     6

     

     

     

     6 well

     

     

     

     9.5

     

     

     

     1 ± 0.1

     

     

     

     1 ± 0.1

     

     

     

     1 ± 0.1

     

     

     

     3

     

     

     

    Note: For more information on enzymatic dissociation and subculturing of cell lines consult Chapter 13 in Culture of Animal Cells, a manual of Basic Technique by R. Ian Freshney, 5th edition, published by Alan R. Liss, N.Y., 2005.  

    Subcultivation Ratio: 1:5 to 1:8

    Cryopreservation
    Complete growth medium, supplemented with an additional 40% FBS and 10% DMSO(v/v)
    Cell culture tested DMSO is available as ATCC Catalog No. 4-X.
    Culture Conditions
    Atmosphere: Air, 95%; carbon dioxide (CO2), 5%
    Temperature: 37°C
    Name of Depositor ATCC
    Year of Origin January 22, 2003
    References

    Caputo, J. L., Biosafety procedures in cell culture. J. Tissue Culture Methods 11:223-227, 1988.

    Hay RJ, Caputo JL, Macy, ML, Eds. (1992) ATCC Quality Control Methods for Cell Lines. 2nd edition, Published by ATCC.

    Fleming, D.O., Richardson, J. H., Tulis, J.J. and Vesley, D., (1995) Laboratory Safety: Principles and Practice. Second edition, ASM press, Washington, DC.

    Freshney RI. Culture of Animal Cells: A Manual of Basic Technique, 5th edition. New York: Wiley Liss; 2005. For more information on enzymatic dissociation and subculturing of cell lines see Chapter 13.

    Nagy A, et al. Manipulating The Mouse Embryo: A Laboratory Manual. Third Edition: Cold Spring Harbor Press; 2003.

    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
    References

    Caputo, J. L., Biosafety procedures in cell culture. J. Tissue Culture Methods 11:223-227, 1988.

    Hay RJ, Caputo JL, Macy, ML, Eds. (1992) ATCC Quality Control Methods for Cell Lines. 2nd edition, Published by ATCC.

    Fleming, D.O., Richardson, J. H., Tulis, J.J. and Vesley, D., (1995) Laboratory Safety: Principles and Practice. Second edition, ASM press, Washington, DC.

    Freshney RI. Culture of Animal Cells: A Manual of Basic Technique, 5th edition. New York: Wiley Liss; 2005. For more information on enzymatic dissociation and subculturing of cell lines see Chapter 13.

    Nagy A, et al. Manipulating The Mouse Embryo: A Laboratory Manual. Third Edition: Cold Spring Harbor Press; 2003.