Fcasjazz

The following antibodies in 1:200 dilutions were used: biotinylated and FITC conjugated CD31, CD45, and TER119 (BD PharMingen; 553371; 553078; 553672; 553372; 553080; 557915); CD24-PE/cy7, CD29-APC (Biolegend; 101–822; 102216) Sca1-PE and Streptavidin-V450 (eBioscience; 12-5981-82; 48-4317-82). Antibody incubation was performed on ice for 25 min in PBS with 5% FBS. All sorting experiments were performed using a FCAS Jazz (Becton Dickinson). The purity of sorted population was routinely checked and ensured to be >95%.

Mammary glands from 8- to 12-wk-old virgin female mice or mice at lactation day 2 were isolated. The minced tissue was digested in RPMI 1640 with 25 mM HEPES, 5% FBS, 1% PSQ, 300U/mL Collagenase III (Worthington) for 2 hours at 37°C. After lysis of the red blood cells, single cell suspension was obtained by sequential incubation with 0.25% Trypsin-EDTA for 5 min and 0.1 mg/mL DNase I (Sigma) for 5 min at 37°C with gentle pipetting, followed by filtration through 40-μm cell strainers. The antibodies used for flow cytometry were: PE-conjugated CD31, CD45, TER119 (BD PharMingen), CD24-PE/cy7 and CD29-APC (Biolegend). All sorting was performed using FACSAria or FCASJazz (Becton Dickinson). The purity of sorted population was routinely checked and ensured to be >95%. Cells were harvested for quantitative RT-PCR experiments. For insulin treatment experiment, primary mammary epithelial cells were seeded in DMEM (Invitrogen) supplemented with 10% FBS. Adhered cells were treated with 10μg/mL insulin for 24 hours and were harvested for quantitative RT-PCR experiments. Cytosolic triacylglycerol and nonesterified fatty acid in primary mammary epithelial cells were quantified following manufacturer's protocols (Triglycerides kit, Shanghai KEHUA bio-engineering CO., and LabAssay NEFA kit, Wako).