Mammosphere Cultivation and Analysis of Luminal and Basal Cells

Freshly sorted LCs (1000 cells) were resuspended in culture medium [DMEM/F12 lacking Phenol Red supplemented with 5 µg/ml insulin (Sigma), 10 ng/ml EGF (Sigma), 100 ng/ml cholera toxin (Sigma) and 5% FCS] and seeded onto 24-well plates in the presence of 5000 irradiated NIH-3T3 cells, as previously described (Sleeman et al., 2007 (link)). Five days later, colonies were fixed with 4% PFA, stained with Hematoxylin and Eosin, and counted.
For three-dimensional mammosphere assays, FACS-sorted luminal or basal cells (10,000 cells) were resuspended in culture medium [DMEM-F12 lacking phenol red supplemented with B27 (1×, Gibco), 20 ng/ml EGF (Sigma), 20 ng/ml bFGF (Gibco), 4 µg/ml heparin (Sigma), 10 µg/ml insulin (Sigma) containing 4% Matrigel] as previously described (Dontu et al., 2003 (link); Spike et al., 2012 (link)). After 15 days in culture, mammospheres were imaged using a stereomicroscope (Nikon SZM800). Three independent experiments were analyzed. For each traced organoid, the size and number of clones were measured using ImageJ software (NIH). For serial passaging, mammospheres were collected by centrifugation and incubated with 0.05% trypsin/EDTA (Gibco) to obtain a single cell suspension. Cells were replated in 4% Matrigel (BD Pharmingen) at a density of 5000 cells/ml, as described above. All cultures were maintained in a 5% CO₂ atmosphere at 37°C.