Normal human breast tissue contains four distinct epithelial subtypes. (a) Freshly dissociated breast epithelial cells from reduction mammoplasty can be divided into four epithelial differentiation states. Primary breast epithelial cells (n = 12) were isolated, lineage depleted, stained with EpCAM, CD24, and CD49f, and quantified as described in Materials and methods. Representative dot plots of EpCAM vs. CD49f staining (left) and CD24 vs. CD49f staining in EpCAM/CD49f populations (middle) are shown. Quantification of CD24 staining in Luminal 1/2, Basal and Mesenchymal populations from 12 patient samples (right, mean ± S.E.). Quantification of immunofluorescence from a representative patient sample is shown (% of total DAPI stained cells, minimum 50 cells analyzed). Luminal 1 and Luminal 2 cells from reduction mammoplasty tissue are predominantly CK 8/18 positive. Basal and Mesenchymal cells expressed CK14, VIM, and αSMA. (b) Freshly dissociated epithelial cells from reduction mammoplasty tissue were stained for EpCAM and CD49f expression, sorted by FACS and cytospun on onto slides for characterization of lineage markers by immunofluorescence. Cytokeratin (CK) 18, 14, smooth muscle actin (αSMA) and vimentin (VIM) immunofluorescence staining and quantification of sorted populations indicates luminal and basal/myoepithelial cell enrichment.