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
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.
Remove and discard culture medium.
Briefly rinse the cell layer with 1XPBS (SCRR-2201) solution to remove all traces of serum, which contain trypsin inhibitor.
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.
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.
Transfer all cells into a centrifuge bottle or tube and centrifuge at 270 x g for 5 minutes.
Remove and discard the supernatant
Add 10 mL complete growth medium to the cell pellet and with 10 mL pipette resuspend the cells gently (create a single-cell suspension).
Add more complete growth medium (Table 1) to the cell suspension as needed to plate cells at approximately 0.8 X 104 cells/cm2.
Place flasks in the incubator @ 37°C with a 5% CO2 in air atmosphere
