Gene expression profile of uniparous mouse mammary cell luminal progenitor subpopulation FACS-sorted based on expression of CD24CD29CD61

We modeled two types of breastfeeding behaviors in wild type FVB/N mice: (1) normal or gradual involution of breast tissue following prolonged breastfeeding and (2) forced or abrupt involution following short-term breastfeeding. To accomplish this, pups were gradually weaned between 28 and 31 days (gradual involution) or abruptly at 7 days postpartum (abrupt involution). FACS was used to relatively quantify mammary epithelial subpopulations. Gene set enrichment analysis was used to analyze gene expression data from mouse mammary luminal progenitor cells. Briefly, single cell suspension of the mammary glands was enriched for lineage-negative (Lin−) epithelial cells excluding the Lin+ cells, specifically hematopoietic (biotinylated CD45 and TER119), endothelial (biotinylated CD31), and immune (biotinylated BP-1) cells. The negatively selected cell population was enriched for luminal epithelium (LE) and mammary stem cell (MaSC)-enriched/basal epithelium (MaSC-enriched). For FACS analysis, Lin− populations were labeled with CD24-PE, CD29-FITC, and CD61-APC while their respective isotypes were used as negative controls. Cell suspensions were incubated with the appropriate antibodies for 30 min on ice. Cells were resuspended in FACS buffer (1 mM EDTA, 1% HI-FBS, and 25 mM HEPES, pH 7.0 in 1× phosphate-buffered saline) and sorted on a FACS BD LSR II Flow Cytometer. 2-3 mice mammary LP tissue isolated from day 28 timepoint were pooled and used for a total of 4 suspensions, 2 AI and 2 GI. P is for AI-LP1, K is for AI-LP2, I is for GI-LP1, and O for GI-LP2. At least 6 mammary glands per mouse were utilized for each replicate.