Anti cd49f fitc

Mouse mammary tissues were dissociated using Collagenase/Hyaluronidase solution (StemCell Technologies) and single cell suspensions were generated according to manufacturer's instructions. Lineage-depleted mammary epithelial cells were prepared using an EasySep mouse mammary stem cell enrichment kit (StemCell Technologies). Flow cytometry was performed by standard procedures using anti-mouse CD24-PE and anti-CD49f-FITC (StemCell Technologies). Fluorescence was recorded using Gallios Flow Cytometer (Beckman Coulter) and analyzed with Kaluza flow cytometry analysis software. Mammary tissues from three animals per genotype were analyzed with similar results. Percentages of stem cell-enriched cell populations in each genotype were quantitated. Lineage-depleted mammary epithelial cells were cultured for mammosphere formation for 7 days as previously described [30] (link).

CD45⁺/TER119⁺, and CD31⁺ cells were removed from dissociated cells using the EasySep biotin selection kit (StemCell Technologies, Vancouver, BC, Canada) according to the manufacturer's instructions. Cells were resuspended in HBSS (Invitrogen) with 2% FBS and incubated with anti-CD24-PE (10 μl/test, Mouse mammary cell enrichment kit, StemCell Technologies), anti-CD49f- FITC (10 μl/test, Mouse mammary cell enrichment kit, StemCell Technologies) or anti-Sca1-APC (0.06 μg/test, eBioscience, San Diego, CA, USA) for 30 min on ice, followed by washing and resuspending in HBSS supplemented with 2% FBS. Analysis and sorting were performed by using a FACSVantage TurboSort SE (BD Biosciences). The data were analyzed using BD FACS Sortware sorter software. For analysis of ERα expression in Sca1⁺CD24^highCD49f⁺ and Sca1⁻CD24^highCD49f⁺ luminal cell populations, sorted cells from Tip30^−/− virgin mice (n=6) were collected and allowed to adhere to a collagen coated 96-well plate before fixation and then analyzed by immunocytochemistry with anti- ERα (MC-20, 1 : 50; Santa Cruz Biotechnology) and inverted microscopy (Nikon Eclipse Ti, Melville, NY, USA).