Breast cancer is a heterogeneous disease, with gene expression profiling describing several molecular subtypes. It has been postulated that these different subtypes may originate through a cell-type specific mechanism of tumourigenesis. Indeed, multiple strands of evidence suggest luminal and basal cells have distinct biologies, which has led us to hypothesize that the different subpopulations within the breast differentiation hierarchy have different susceptibilities to acquiring and propagating genomic imbalance due to different inherent responses to DNA damage. Our data from colony forming cell assays revealed similar survival sensitivities to increasing radiation dose within luminal-restricted-, myoepithelial-restricted- and bi-potent progenitors from fresh dissociations of normal breast tissues. To further explore the molecular and cellular basis of DNA damage response in mixed populations of breast epithelial cells, we developed a high-content screening protocol to monitor the response in the different subpopulations after treatment with increasing doses of ionizing radiation. Normal human breast epithelial cells from reduction mammoplasties cultured in multi-well format were stained using a 4-colour immunofluorescence technique for cytokeratin 14 and 19 to detect myoepithelial and luminal cells respectively, γH2AX to identify nuclear foci representing the initiation of DNA damage response, and DAPI to examine total cell numbers and generate cell cycle profile data stratified by cell type. Imaging and image analysis were performed using the GE IN Cell Analyzer 2000 in combination with its analysis software. To date, data from n=3 patients indicate heightened sensitivity and differential G2M accumulation in CK19+ luminal cells and the mechanisms underlying this are being explored. This represents the first high-throughput, high content method to detect DNA damage response in distinct breast epithelial cell types in mixed adherent cultures. We also present extensive validation data demonstrating how this powerful screening method can be used in several other applications to stratify cell-type specific responses in mixed cultures.