At clonal cell numbers, the outgrowth potential of LIN?CD24+CD29H cells was significantly increased (= .036) as compared with cells >10 m in size (Table 1). with lineage-negative (LIN?) control cells. Limiting dilution transplantation assays indicated that the repopulating ability of LIN?CD24+CD29H cells that were >10 m in size was significantly increased as compared with cells marked by CD24 and CD29 alone. These results suggest that MaSCs can be further isolated by sorting based on size/FSC. These findings have critical implications for understanding mammary gland stem cell biology, an important requisite step for understanding the etiology of breast cancer. with approval from the Baylor College of Medicine Institutional Animal Care and Use Committee. Mammary epithelial cells (MECs) were derived from freshly dissected thoracic and inguinal (without the lymph node) mammary glands of 8C12-week old female mice (FVB.Cg-Tg(CAG-EGFP)B5Nagy/J; Jackson Laboratory, Bar Harbor, ME, http://www.jax.org). Glands were minced into fragments (<1 mm) using a razor blade and digested in Dulbecco's modified Eagle's medium (DMEM)/F12 medium containing 1 mg/ml MLN4924 (Pevonedistat) collagenase A (Roche Applied Science, Indianapolis, IN, https://www.roche-applied-science.com), 100 U/ml hyaluronidase (Sigma-Aldrich, St. Louis, MO, http://www.sigmaaldrich.com), and 1 antibiotic-antimycotic (Invitrogen, Carlsbad, CA, http://www.invitrogen.com) for 14 hours at 37C with shaking at 75 rpm. Cells were washed three times with 1 phosphate-buffered saline (PBS) containing 5% fetal bovine serum (FBS) and incubated with 0.25% trypsin-EDTA at room temperature for 2C3 minutes with rocking. Trypsin was inactivated with 1 PBS containing 5% FBS; cells were centrifuged and filtered through a 40-m cell strainer. Single cells were counted on a hemacytometer using trypan blue. Fluorescence-Activated Cell Sorting Freshly isolated MECs were resuspended at a concentration of 1 1 107 cells per ml in Hanks' balanced saline solution (HBSS) containing 2% FBS and 100 mM Hepes (HBSS+), and stained with specific antibodies as previously described . A complete list of antibodies is provided in supplemental online Table 1. Cells were filtered through a 40-m cell strainer, incubated with a dead cell exclusion dye (Sytox red/blue; Invitrogen), and sorted on a FACSAria II Cell Sorting Flow Cytometer (BD Biosciences, San Jose, CA, http://www.bdbiosciences.com). Prior to sorting, sizing beads (SPERO Particle Size Standard Kit; Spherotech, Inc., Lake Forest, IL, http://www.spherotech.com) were analyzed to determine estimated sizes of MECs. For transplantation assays, cells were sorted into DMEM/F12 medium containing 5% FBS, 5 g/ml insulin, 1 g/ml hydrocortisone, 10 ng/ml epidermal growth factor (EGF), and 1 antibiotic-antimycotic. A postsort analysis was performed to assess the purity of the sorted cell populations and was estimated (from four independent experiments) to be 97.9 0.5% for LIN? cells, 81.6 2.6% for cells >10 m, 91.2 0.9% for LIN?CD24+CD29H cells, and 92.4 1.5% for LIN?CD24+CD29H cells >10 m. Data were analyzed using FlowJo 2 v9.5.2 (Tree Star, Ashland, OR, http://www.treestar.com). Mammosphere MLN4924 (Pevonedistat) Assays MECs were FACS-sorted VAV2 based on size into DMEM/F12 media containing 20 ng/ml EGF, 20 ng/ml basic fibroblast growth factor, B27, and 1 antibiotic-antimycotic (MS media). Cells were washed, resuspended in MS media at a concentration of 15,000 cells per ml, and plated in ultralow-attachment plates (2 ml per well). Cells were fed every 4 days for 12 days, at which time they were dissociated as previously described . Secondary mammospheres were cultured for an additional 14 days as previously described . Twelve wells for each group were counted and the percentage of mammosphere forming cells was calculated as a measure of mammosphere efficiency. Transplantation Assays and Whole Mount Analysis FACS-sorted cell subpopulations were washed with 1 PBS and resuspended in a 1:1 solution of PBS and Matrigel (BD Biosciences). Cells were serially diluted for limited dilution transplantation assays. Inguinal glands of recipient female mice (FvB/NJ; Jackson Laboratory) were cleared MLN4924 (Pevonedistat) at 3 weeks of age, and cells were transplanted 2C3 weeks later. Ten microliters of cells was injected into contralateral cleared fat pads using a 26-gauge needle and 50-l Hamilton glass syringe . Animals were sacrificed 8 weeks after transplantation, and contralateral mammary glands were removed, compressed between two glass slides, and visualized using a Leica MZ16F fluorescent stereoscope (Leica Microsystems, Buffalo Grove, IL, http://www.leica-microsystems.com). Mammary glands showing at least 5% fat pad filled were included in the analysis. For glands that displayed no outgrowth, the lack of epithelium was verified by neutral red staining, and these were included in the calculation of take rate. For neutral red staining, contralateral mammary glands were fixed in ice-cold 4% paraformaldehyde for 1 hour and stained as previously described . Statistical Analysis Data from mammosphere assays are presented as the means SEM. Significance values were calculated with MLN4924 (Pevonedistat) a one-way analysis of variance model followed by Bonferroni pairwise MLN4924 (Pevonedistat) comparisons between groups. Limiting dilution transplantation results were analyzed by.