Mammary Epithelial Cell Growth Medium

Protocol tips

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	Seed of single primary mammary epithelial cells at limiting dilutions (100 cells/ml) Use of 96-well ultra-low-attachment plates Fix, stain and count colonies according to size and morphological criteria

Protocol tips
Seed of single primary mammary epithelial cells at limiting dilutions (100 cells/ml) Use of 96-well ultra-low-attachment plates Fix, stain and count colonies according to size and morphological criteria

Publication protocol

For mammosphere assays, single primary mammary epithelial cells were seeded at limiting dilutions (100 cells/ml) on 96-well ultra-low-attachment plates (BD Corning) in serum-free basal mammary epithelial growth medium (Promocell) supplemented with B27, 4 μg/ml heparin, 20 ng/ml basic fibroblast growth factor (both Life Technologies) and epithelial growth factor (EGF) (Sigma). For E-CFC, primary cells were seeded in complete Epicult-B medium (STEMCELL Technologies). Colonies were fixed, stained, and counted according to size and morphological criteria. Early, highly proliferative E-CFCs were considered to give rise to large colonies and late, slowly proliferating E-CFC produced small colonies, measured by Ki67 staining. Lineage properties of colonies are defined using classical criteria and based on keratin 14 and keratin 18 staining Quantification of E-CFC following primary mammosphere formation and dissociation reveals the initial progenitor content that is rapidly exhausted by in vitro culture. In contrast, performing the E-CFC assay using cells obtained from sequential spheres (secondary, tertiary, or more in some cases) allows quantification of epithelial colonies derived from early common progenitors that have differentiated during prolonged culture to generate new progenitors and tested in the ECP-DC assay.

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