This cell line can be used to study normal epithelial cell function and cell transformation.

EpH4-Ev was produced by transfecting parental EpH4, an immortalized mouse mammary epithelial cell line, with an empty expression vector carrying a puromycin resistance gene. xa0Stable clones were isolated by selection in medium containing 1.2 mg/mL puromycin.

The EpH4 cell line presents an attractive model in which to study transformation because the cells exhibit morphological and functional characteristics typical of normal mammary epithelial cells. This cell line has been used to generate constitutively activated MEK1 transformed cell line B-MEKDD 116 (ATCC CRL-3069). There are three additional related cell lines: EpH 1424 (ATCC CRL-3071), EpH4 1424.1 (ATCC CRL-3209), EpH4 1424.2 (ATCC CRL-3210).

• 10% Bovine Calf Serum
• 1.2 mcg/mL Puromycin

1. Remove and discard culture medium.
2. Briefly rinse the cell layer with Ca++/Mg++ free Dulbeccos 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 4 x 10³ to 8 x 10³ 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.

Fantin VR, et al. A novel mitochondriotoxic small molecule that selectively inhibits tumor cell growth. Cancer Cell 2(1): 29-42, 2002 PubMed: 12150823