MEBMTM Mammary Epithelial Cell Growth Basal Medium

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	Culture isolated MECs in MEC culture medium for 1 day, before mammosphere culture Seed single MECs at a density of 25 000 viable cells/ml Use of ultralow attachment plates

Protocol tips
Culture isolated MECs in MEC culture medium for 1 day, before mammosphere culture Seed single MECs at a density of 25 000 viable cells/ml Use of ultralow attachment plates

Publication protocol

MECs were isolated as described earlier.15 Breast glands were dissected from 8-week-old female mice and minced with a razor blade. The tissue was placed in culture medium (DMEM/F12 with 1 mM glutamine, 5 μg/ml insulin, 500 ng/ml hydrocortisone, 10 ng/ml epidermal growth factor and 20 ng/ml cholera toxin) supplemented with 5% fetal bovine serum (Hyclone, South Logan, UT, USA) and containing 300 U/ml collagenase IV (Invitrogen, Grand Island, NY, USA) and 100 U/ml hyaluronidase (Sigma, St Louis, MO, USA), and digested for 1 h at 37 °C. The resultant organoid suspension was sequentially digested in 0.25% trypsin-EGTA for 2 min, 5 mg/ml dispase (Stemcell Technologies, Vancouver, Canada) and 0.1 mg/ml DNase (Stemcell Technologies) for 5 min and 0.64% NH4Cl for 3 min before filtration through a 40-μm cell strainer. The filtrated cells ware cultured or labeled. The cell culture medium was used as described earlier.36Cells were cultured in complete growth media containing 5 μg/ml insulin, 1 μg/ml hydrocortisone (Sigma), 3 ng/ml epidermal growth factor, 10% fetal bovine serum and 50 U/ml penicillin/streptomycin in DMEM/F12 (Invitrogen). All cultures were maintained at 37 °C in a 5% CO2 atmosphere. For mammosphere culture, the isolated MECs were cultured in MEC culture medium for 1 day. Single cells were plated in ultralow attachment plates (Corning, Corning, NY, USA) at a density of 25 000 viable cells/ml in primary culture. Cells were grown in a serum-free mammary epithelial growth medium (Lonza, Walkersville, MD, USA), supplemented with B27 (Invitrogen), 20 ng/ml epidermal growth factor (Sigma), 20 ng/ml bFGF (Invitrogen) and 4 μg/ml heparin (Sigma). Half of the medium was changed every 3 days. The mammospheres were cultured for 20 days, and the ones with a diameter >50 μm were counted.

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Manufacturer protocol

Download the product protocol from Lonza for MEBMTM Mammary Epithelial Cell Growth Basal Medium below.

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