ence kit. Flow Cytometry Cells were cultured accordingly then washed twice with PBS and detached using 2 mM EDTA in PBS at 37uC for 5 min. For flow cytometric analysis, all subsequent incubation steps were performed on ice and centrifugation steps performed at 4uC. For analysis of Apo2L/TRAIL receptor expression, cells were resuspended in ice cold PBS and centrifuged for 6 min at 12,000 rpm. Cells were resuspended in ice cold PBS and PBS/ Azide solution and centrifuged at 12,000 rpm, 5 min. Cells were resuspended at 26106 cells/ml in blocking buffer. Monoclonal antibodies or the isotype-matched nonbinding control antibodies were diluted in blocking buffer to 10 mg/ml, added to 50 ml aliquots of cell suspension and incubated for 45 min. Cells were then washed 12747794 twice with 2 ml of wash buffer and collected by centrifugation. PEconjugated antibody was added to the resuspended cell pellets, diluted 1/50 in wash buffer. The cells were incubated for a further 45 min in the dark, washed three times as above, then resuspended and fixed in 0.3 ml ice-cold 1% w/v paraformaldehyde for analysis. For analysis of cell cycle, cells were cultured for 24 h, then serum starved for a further 24 h and media was replaced with growth media. At the appropriate time point cells were detached as above and collected by centrifugation. Cells were washed in ice cold PBS and centrifuged. Cells were then resuspended in 200 ml PBS +0.1% FBS, and fixed in ice cold 70% ethanol then incubated for 1 h at 4uC. Cells were washed as above and resuspended in 1 ml of solution containing; 0.1% Triton-X 100, 200 mg/ml RNAse free and 40 mg/ml propidium iodide, incubated for 20 min at 37uC then analysed. Materials and Methods Cells and Reagents MCF-7 and MDA-MB-231 human breast carcinoma cell lines were obtained from the American Type Culture Collection. Cells were cultured in RPMI 1640, supplemented with 10% foetal bovine serum, glutamine, HEPES penicillin-streptomycin and minocyclin , in a humidified atmosphere containing 5% CO2. MCF-7 PTHrP overexpressing cells are as previously described. Human recombinant Apo2L/TRAIL was obtained from Preprotech. Monoclonal antibodies against human Apo2L/TRAIL-R1/DR4, Apo2L/ TRAIL-R2/DR5, Apo2L/TRAIL-R3/DcR1, Apo2L/TRAIL-R4, goat polyclonal antibodies against human Apo2L/TRAIL-R1/DR4, Apo2L/TRAIL-R2/DR5, Apo2L/TRAIL-R3/DcR1, Apo2L/TRAIL-R4, mouse IgG2B, mouse IgG1, monoclonal antihuman Caspase-7, Caspase-8, Caspase-9, Caspase-10 were from R&D Systems. Polyclonal antihuman Caspase-6 was purchased from Cell 10914735 Signalling Technology. Monoclonal b-actin antibody was from Sigma-Aldrich. Monoclonal PARP antibody was from Trevigen. Goat anti-mouse HRP was from Cell Signalling. Goat anti-rabbit-HRP was from Dako Cytomation. Apoptotic DNA Laddering Assay Cells were cultured and treated with 100 ng/ml recombinant Apo2L/TRAIL or left untreated with 24 h. DNA was isolated and treated using the Apoptotic DNA Ladder Kit according to the manufacture’s protocol. Measurement of Cell Viability For determination of Apo2L/TRAIL mediated cytotoxicity, 16104 cells per well were seeded into 96-well microtiter plates and allowed to adhere to the plate for 24 h. Cells were treated according and/or then treated with 100 ng/ml Apo2L/TRAIL for 24 h. Cell viability was determined by staining the cells with crystal AZ-6102 site violet and measuring the OD570 of cell lysates. DAPI staining of nuclei- Cells were seeded on plastic chamber slides and stimulated as indicated. After 2 washes wi