B-MEKDD 116 - CRL-3069 | ATCC (2024)

B-MEKDD 116 was produced by transfecting parental EpH4, a mouse mammary epithelial cell line, with an expression vector containing Glu-Glu epitope-tagged phosphorylation site MEK1 mutant (MEKDD). After selection in complete medium supplemented with 1 mg/mL G418 for 14 days, individual clones were isolated and expanded.

B-MEKDD 116 was produced by transfecting parental EpH4, a mouse mammary epithelial cell line, with an expression vector containing Glu-Glu epitope-tagged phosphorylation site MEK1 mutant (MEKDD). After selection in complete medium supplemented with 1 mg/mL G418 for 14 days, individual clones were isolated and expanded.

This cell line stably expresses constitutively activated form of MEK1 (Asp218/Asp222 MEK1 phosphorylation site mutant), and can be used in MEK-MAPK pathway studies. This cell line is tumorigenic, and can be used to build a breast cancer in vivomodel. There are four additional related cell lines: EpH 1424 cell line (ATCC CRL-3071), EpH4 1424.1 cell line (ATCC CRL-3209), EpH4 1424.2 cell line (ATCC CRL-3210), and EpH4-Ev cell line (ATCC CRL-3063).

The B-MEKDD 116 cell line produces the constitutively activated MEK1 mutant MEKDD which has been tagged with Glu-Glu, verified at ATCC.

1. Check all containers for leakage or breakage.
2. Remove the frozen cells from the dry ice packaging and immediately place the cells at a temperature below ­-130°C, preferably in liquid nitrogen vapor, until ready for use.

37°C

95% Air, 5% CO₂

To insure the highest level of viability, thaw the vial and initiate the culture as soon as possible upon receipt. If upon arrival, continued storage of the frozen culture is necessary, it should be stored in liquid nitrogen vapor phase and not at –70°C. Storage at –70°C will result in loss of viability.

1. Thaw the vial by gentle agitation in a 37°C water bath. To reduce the possibility of contamination, keep the O-ring and cap out of the water. Thawing should be rapid (approximately 2 minutes).
2. Remove the vial from the water bath as soon as the contents are thawed, and decontaminate by dipping in or spraying with 70% ethanol. All of the operations from this point on should be carried out under strict aseptic conditions.
3. Transfer the vial contents to a centrifuge tube containing 9.0 mL complete culture medium. and spin at approximately 125 x g for 5 to 7 minutes.
4. Resuspend cell pellet with the recommended complete medium (see the specific batch information for the culture recommended dilution ratio). and dispense into a 25 cm² or a 75 cm² culture flask. It is important to avoid excessive alkalinity of the medium during recovery of the cells. It is suggested that, prior to the addition of the vial contents, the culture vessel containing the complete growth medium be placed into the incubator for at least 15 minutes to allow the medium to reach its normal pH (7.0 to 7.6).
5. Incubate the culture at 37°C in a suitable incubator. A 5% CO₂ in air atmosphere is recommended if using the medium described on this product sheet.

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 1.0 to 2.0 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. An inoculum of 8 x 10³ to 1.5 x 10⁴ viable cells/cm² is recommended.
6. Incubate cultures at 37°C. Subculture when the cell concentration is between 8 x 10⁴ to 1.5 x 10⁵ cells/cm².

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