An improved definition of mouse mammary epithelial side population cells.

Abstract

Mammary epithelial side population cells have been suggested as candidate mammary stem cells. To date, for technical reasons, these cells have been poorly defined and cross-comparison of data between different laboratories has been difficult. Here, we set out to define mammary side population cells in a way that improves the ability to carry out such comparisons. Mouse mammary epithelial cells were stained with Hoechst 33342. Light scatter, PI staining and clonogenicity of different regions of the Hoechst profile were examined. Time-course analyzes of Hoechst 33342 loading were carried out. Detailed examination of the light scatter and PI staining of Hoechst 33342-stained mammary cells enabled single live side population and non-side population cells to be defined with greater accuracy. Comparison of ABC pump inhibitors identified potential discrepancies in results obtained using these inhibitors. Time-course analyzes enabled side populations cells to be identified as a dynamic cell population that could be defined accurately by using the relationship between Hoechst 33342-staining profiles of consecutive time points. Defining the side population of solid tissues as a 'stabilized side population percentage' will enable a more rigorous study of the side population phenomenon and improve evaluation of results from different laboratories.