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• 20 ng/ml epidermal growth factor (Do not filter)
• 100 ng/ml cholera toxin
• 0.01 mg/ml bovine insulin
• 500 ng/ml hydrocortisone
• 5% horse serum

Volumes used in this protocol are for 75 cm² flask; 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 0.25% (w/v) Trypsin-0.03% (w/v) 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. Incubate cultures at 37°C.

Soule H, McGrath CM. Immortal human mammary epithelial cell lines. US Patent 5,026,637 dated Jun 25 1991

Pauley RJ, et al. Immortal human mammary epithelial cell sublines. US Patent 5,206,165 dated Apr 27 1993

Pauley RJ, et al. Immortal human mammary epithelial cell sublines. US Patent 5,238,840 dated Aug 24 1993

Soule HD, McGrath CM. A simplified method for passage and long-term growth of human mammary epithelial cells. In Vitro Cell. Dev. Biol. 22: 6-12, 1986. PubMed: 2418007

Soule HD, et al. Isolation and characterization of a spontaneously immortalized human breast epithelial cell line, MCF-10. Cancer Res. 50: 6075-6086, 1990. PubMed: 1975513

Tait L, et al. Ultrastructural and immunocytochemical characterization of an immortalized human breast epithelial cell line, MCF-10. Cancer Res. 50: 6087-6094, 1990. PubMed: 1697506