Fig 2: ERpos cells are purified and tracked by sequential CD326/CD271–CD166/CD117 FACS followed by multicolour staining and qRT–PCR.(a) Multicolour flow cytometry of uncultured HBECs incubated with CD326/CD271/CD166/CD117 and visualized pairwise (left diagrams) to recover luminal cells (CD326high) and basal cells (CD271high) and from the luminal gate CD166high and CD117high cells. Smears of sorted cells were stained (right panel) with either of the markers against basal cells, cytokeratin K14; luminal cells, cytokeratin K18; luminal progenitors, cytokeratin K15; Ks20.8 or ER–PR and counterstained with DAPI nuclear stain. Hormone receptor-positive cells are observed primarily among CD166high cells. Scale bar, 50 µm. (b) Purity of sorted cells as determined by staining of smears followed by quantification of the percentage of cells stained with either of the markers cytokeratin K14, K18, K15, Ks20.8 or hormone receptors (ER–PR; 3 × 100 cells per slide, error bars indicate s.d.'s). (c) Heatmap representing qRT–PCR analysis of the relative gene expression of lineage markers in sorted basal cells (basal), CD117high luminal cells (CD117) and CD166high luminal cells (CD166) from six different biopsies. Data confirm lineage-specific transcriptional profiles of the three cell populations and restricts ER expression (ESR1) primarily to CD166high luminal cells. Colour bar indicates the fold difference of the relative gene expression in log2 scale.