Breast cell lines MCF10A

and MDA-MB-231 cells (ATCC) grown normally in DMEM-F12, 5% horse serum, 0.5 μg/ml hydrocortisone, 10 μg/ml insulin, 100 ng/ml cholera toxin, 20 ng/ml human recombinant EGF (MCF10A) or DMEM, 10% FBS, 2 mM L-glutamine(MDA-MB-231) were conditioned in MEGM for 2-3 weeks and used in flow cytometry experiments as controls for normal and tumourogenic phenotypes respectively. Proliferation assays Primary find more cells (5 × 103) were plated in triplicate and harvested after 0, 3 or 6 days. Cyquant solution was incubated on freeze-thawed cells (5 min), and emitted fluorescence detected at 520 nm on a Wallac plate-reader. Fluorescence readings of unknown samples were translated into cell numbers by referring to two separate fluorescence standard curves – one for non-tumour and one for tumour cultures- constructed from known cell numbers (Additional file 2). The slope of each proliferation graph was calculated from the linear regression line using the formula y = mx+c, where m = slope and c = y-intercept. Senescence-associated β-galactosidase P505-15 supplier assays Primary

cells (5 × 104) were plated in duplicate, and stained for senescence-associated β-galactosidase activity [9]. Three brightfield micrographs per condition were captured, and blue senescent cells GF120918 mouse expressed as a percentage of total cells/field. Immunofluorescence staining for epithelial and myoepithelial markers Primary cells (passage 1-2) grown in chamber slides were fixed in 3.7% paraformaldehyde and immunostained for epithelial (K19, K18, ESA) or myoepithelial many (SMA, K14, VIM) markers using DAPI as a nuclear counter-stain. Primary antibodies were omitted in negative controls, and slides visualized on a Zeiss LSM510-meta confocal microscope. SDS-PAGE and Western blotting Confluent primary cultures were harvested in RIPA (20 mM Tris-HCl pH7.5, 150 mM NaCl, 5 mM EDTA, 1% Triton-X100) containing protease and phosphatase inhibitors. Lysates were dounced and 25 μg supernatant subjected

to SDS-PAGE and Western blot analysis for K19, K18, VIM and p63. FACS analysis of putative progenitor cell populations Confluent passage 0 primary cells (T25 flask/condition) were trypsinized, blocked in human serum and co-incubated with FITC-conjugated mouse anti-human EPCAM and PE-conjugated mouse anti-human CALLA (4°C/30 min). Negative controls were unlabelled or single-stained with FITC-EPCAM, PE-CALLA, FITC-IgG or PE-IgG. Cells were analyzed on a Beckman Coulter Cyan-ADP and/or an Accuri-C6 flow cytometer. Cells were sorted into CALLA+/EPCAM+, CALLA+/EPCAM-, CALLA-/EPCAM- or CALLA-/EPCAM+ populations on a BD FACSAria cell sorter. Some passage 0 cells were analyzed for activity of the stem cell marker ALDH by Aldefluor assay [5]. Briefly, 2 × 105 cells were resuspended in assay buffer and incubated with activated substrate or the negative control reagent before analysis. Transmission electron microscopy (TEM) Passage 0 primary cultures or HMECs were fixed with 2.