Isolation and Culture of Murine Mammary Epithelial Cells

Mammary glands from 8 to 12-week-old virgin female mice were enzymatically digested and single cell suspensions of purified mammary epithelial cells (MECs) were obtained following a standard protocol^{49 (link)}. Briefly, mammary glands were digested at 37 °C for 1–2 h in Epi-Cult-B medium (Stem Cell Technologies Inc) with 600 U/ml collagenase (Sigma Aldrich) and 200 U/ml hyaluronidase (Sigma Aldrich). After lysis of the red blood cells with NH₄Cl, the remaining cells were washed with PBS/0.02% w/v EDTA. Cells were then dissociated with 0.25% w/v trypsin, 0.2% w/v EDTA for 2 min by gentle pipetting, then incubated in 5 mg/ml Dispase II (Sigma Aldrich) plus 1 μg/ml DNase I (Sigma Aldrich) for 5 min, followed by filtration through a 40 μM cell strainer (BD Falcon). MECs were then purified using the EasySep Mouse Mammary Stem Cell Enrichment Kit (Stem Cell Technologies Inc). MECs were seeded on top of 50 or 0.5 kPa Easy Coat hydrogels (Cell guidance system) coated with 10 μg/ml fibronectin and harvested after 24 h.