Mammary Epithelial Cell Growth Medium

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	Use of collagen I gels For detached gels, use siloxane-coated 24-well or 48-well plates For attached gels, use uncoated 24-well plates

Protocol tips
Use of collagen I gels For detached gels, use siloxane-coated 24-well or 48-well plates For attached gels, use uncoated 24-well plates

Publication protocol

Isolation and culture of human mammary epithelial cells: Mammary gland tissue was obtained from healthy women undergoing reduction mammoplasty at the Nymphenburg Clinic for Plastic and Aesthetic Surgery, in accordance with the regulations of the ethics committee of the Ludwig-Maximilian University, Munich, Germany (proposal 397-12). For single-cell suspensions of primary HMECs, the ductal tree was minced into about 1.0 mm3 pieces and digested sequentially using collagenase I together with hyaluronidase (both Sigma), Trypsin-EDTA and dispase (Life Technologies), and then cryopreserved. Before further processing, cells were filtered through a 40 µm strainer, to remove tissue fragments and cell aggregates. Cells were seeded in 2D on polystyrene cell culture plates or in collagen I gels in mammary epithelial cell growth medium (MECGM, PromoCell) supplemented with 1% pen/strep (Invitrogen), 0.5% FCS (Pan Biotech), 3 µM Y-27632 (Biomol) and 10 µM forskolin (Biomol), unless stated otherwise. After an establishment period of 5 days, medium was changed to MECGM supplemented with 1% pen/strep and 10 µM forskolin, unless stated otherwise. 3D collagen I gels: Single-cell suspensions were quickly mixed with neutralizing solution, and acidified rat tail collagen I (Corning) was added, resulting in a final concentration of 1.3 mg/ml. Next, the gel mixture was plated into siloxane-coated 24-well or 48-well plates. After polymerization of the gel, medium with supplements was added and gels were detached from the well. Attached gels were prepared in uncoated 24-well plates.

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Manufacturer protocol

Download the product protocol from PromoCell for Mammary Epithelial Cell Growth Medium below.

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