MEBMTM Mammary Epithelial Cell Growth Basal Medium

Protocol tips

Upstream tips	Protocol tips	Downstream tips
To prevent attachment of cells all dishes/plates, coat them with polyhydroxyethylmethacrylate (PolyHEMA)	Seed HMECs at a density of 10,000 cells/ml and 50,000 cells/ml, respectively, in 3-, 6-, or 10-cm cell culture dishes or 96-well flat-bottom plates

Upstream tips
To prevent attachment of cells all dishes/plates, coat them with polyhydroxyethylmethacrylate (PolyHEMA)
Protocol tips
Seed HMECs at a density of 10,000 cells/ml and 50,000 cells/ml, respectively, in 3-, 6-, or 10-cm cell culture dishes or 96-well flat-bottom plates

Publication protocol

Mammosphere culture: Cell lines and primary HMECs were seeded at a density of 10,000 cells/ml and 50,000 cells/ml, respectively, in 3-, 6-, or 10-cm cell culture dishes or 96-well flat-bottom plates (Thermo Fisher Scientific, Germany; Sigma-Aldrich, Germany; TPP AG, Switzerland). For analyses using the Operetta high-content imaging system cells were plated with 10,000–50,000 cells/ml in 96-well µClear plates (Greiner Bio-One, Germany). To prevent attachment of cells all dishes/plates were coated with polyhydroxyethylmethacrylate (PolyHEMA) (12 mg/ml in 95% ethanol, Sigma-Aldrich, Germany) overnight. PolyHEMA-coated dishes/plates were ultraviolet sterilized for 30 min. Cells were cultured in mammosphere medium consisting of MEBM (mammary epithelial cell basal medium) (Lonza, Germany) supplemented with 1% penicillin/streptomycin (Sigma-Aldrich, Germany), 1×B27 (Life Technologies, Germany), 10 ng/ml EGF (Sigma-Aldrich, Germany), 10 ng/ml basic fibroblast growth factor (bFGF) (Sigma-Aldrich, Germany), 4 µg/ml heparin (Sigma-Aldrich, Germany), and 1% methylcellulose, if the Operetta CLS high-content imaging system was used. For some analyses, mammosphere media were supplemented additionally with 10 ng/ml IL-6 (Sigma-Aldrich, Germany), 1.5 µg/ml anti-IL-6 antibody (Sigma-Aldrich, Germany), 20 ng/ml HIL6, and 0.1 or 10 ng/ml recombinant human sgp130-Fc (R&D Systems, Germany). Mammospheres were cultured in a humidified atmosphere with 5.5% CO2 and 7% O2 at 37 °C for 4 or 7 days. For setting up of secondary mammosphere cultures, conducting flow cytometric or single-cell expression analyses, first-generation mammospheres were collected on day 7 by gentle centrifugation (100 × g), dissociated into single-cell suspension with trypsin-EDTA (Pan-Biotech, Germany) for 3 min, followed by trypsin-neutralizing solution (Lonza, Germany). Single-cell suspensions of secondary mammospheres were obtained as described for day 7 first-generation mammospheres.

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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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