The CD24/Sca1 and CD49fHigh/CD24 flow-cytometry profiles of parous and age-matched virgin mice are similar. (A) Schematic illustration of the cell-isolation strategy and representative flow-cytometry pseudocolor plots of mammary cells from age-matched virgin control mice. After depletion of CD45+ white blood cells, luminal and basal mammary epithelial cells were separated on the basis of CD24 and Sca1 expression. Further separation of basal cells into myoepithelial and basal stem/progenitor cell subpopulations was based on the expression of CD24 and CD49f. The isolated mammary epithelial cell subpopulations included luminal Sca1+ (CD24+HighSca1+) cells, luminal Sca1- (CD24+HighSca1-) cells, basal CD49fHigh (CD24+LowSca1-CD49fHigh) or basal stem/progenitor cells, and basal myoepithelial (CD24+LowSca1-CD49fLow) cells. (B) Outline of the mouse mating, parturition, weaning, and involution protocol. (C) Representative flow-cytometry pseudocolor plots of mammary cells from parous mice. The gates applied were the same as those for age-matched virgin controls. (D) Bar graph showing the distribution of mammary epithelial cell subpopulations comparing cells from parous with age-matched virgin control mice. Data represent the mean ± SEM of seven cell-isolation experiments with a minimum of 10 mice per experiment. The proportion of luminal Sca1+ cells was reduced by approximately 50% in parous mice (P = 0.02 with a two-tailed unpaired Student t test).