title: A protocol to quantify mammary early common progenitors from long-term mammosphere culture.
abstract: Here we describe a protocol established in our laboratory to quantify early common progenitors/stem cells grown as spheres in non-adherent culture conditions. This protocol is based on the combination of two functional tests: the mammosphere assay to maintain and enrich early mammary progenitors/stem cells, and the epithelial colony-forming cells (E-CFC) assay to identify and quantify further differentiated epithelial progenitors. Primary spheres mainly contain progenitors and rare stem/early common progenitor cells while secondary and tertiary spheres contain progenitor cells derived from the early common progenitor/stem cell population maintained through passages and partially differentiated. Spheres are enzymatically and mechanically dissociated; the derived cells are subsequently plated on irradiated NIH-3T3 fibroblasts for further processing, as in the E-CFC assay. The principle of this assay is to quantify the number of epithelial colonies generated by cells present in the different sequential spheres. This assay has therefore been named ECP-DC, standing for Early Common Progenitor-Derived Colonies assay.
authors.affiliation: CNRS UMR5286, Centre de Recherche en Cancérologie de Lyon, F-69000 Lyon, France.
authors.forename: Elodie
authors.initials: E
authors.lastname: Bachelard-Cascales
authors.forename: Marion
authors.initials: M
authors.lastname: Chapellier
authors.forename: Emmanuel
authors.initials: E
authors.lastname: Delay
authors.forename: Véronique
authors.initials: V
authors.lastname: Maguer-Satta
mesh_terms: D000328:Adult; D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods*; D002454:Cell Differentiation; D003114:Colony-Forming Units Assay; D006801:Humans; D042361:Mammary Glands, Human / Q000166:cytology*; D051379:Mice; D041681:NIH 3T3 Cells; D018874:Spheroids, Cellular / Q000166:cytology* / Q000378:metabolism; D013234:Stem Cells / Q000166:cytology* / Q000378:metabolism; D013997:Time Factors