M J Smalley1, I Titley, A Ashworth. 1. The Breakthrough Breast Cancer Research Centre, The Institute of Cancer Research, London, UK.
Abstract
BACKGROUND: 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. METHODS: 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. RESULTS: 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. DISCUSSION: 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.
BACKGROUND: 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. METHODS: 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. RESULTS: 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. DISCUSSION: 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.
Authors: Frances S Kittrell; Martha Z Carletti; Sofia Kerbawy; Jessica Heestand; Wa Xian; Mei Zhang; Heather L Lamarca; Arnoud Sonnenberg; Jeffrey M Rosen; Daniel Medina; Fariba Behbod Journal: Breast Cancer Res Date: 2011-04-05 Impact factor: 6.466
Authors: Katherine E Sleeman; Howard Kendrick; David Robertson; Clare M Isacke; Alan Ashworth; Matthew J Smalley Journal: J Cell Biol Date: 2006-12-26 Impact factor: 10.539
Authors: Annette Meeson; Andrew Fuller; David T Breault; W Andrew Owens; Gavin D Richardson Journal: Stem Cell Rev Rep Date: 2013-10 Impact factor: 5.739