Mouse luminal compartment contains distinct subpopulations. (A) Distribution of epithelial cell adhesion molecule (EpCAM) and CD49f among Lin- cells resolves the luminal and basal cell subpopulations (left). Expression of Sca1 and CD49b among the luminal cells resolves three subpopulations (middle), whose morphology can be visualised using the Image Stream™ analyser (Merck Millipore, Watford, Hertfordshire, UK) (right). (B) Expression of Sca1 and CD49b in pregnant (left; R9 gate) and 6 weeks post involution (right; R7 gate) mammary glands indicating the emergence of a fourth luminal cell population. (C) Bar chart showing the distribution of colony-forming cells (CFCs; top) and cloning efficiencies (middle) of the three luminal populations. Images of CFCs indicating only CD49b+ cells can form colonies (bottom). (D) Immunocytochemical and immunofluorescence analysis of mouse luminal subpopulations. Cells were sorted, fixed onto slides and stained to detect oestrogen receptor (ER), keratin (Krt)18, Krt14 and Krt5. (E) Bar chart showing the percentage positive cells for each population. (F) Gene expression analysis of Sca1-CD49b+ and Sca1+CD49b+ populations relative to the comparator (Sca1+CD49b-) for luminal and basal transcripts. Error bars for all indicate the standard error of the mean for six independent experiments. *P < 0.05, **P < 0.01, ***P < 0.0001. Scale bars = 10 μm.