Annexin 5 fitc apoptosis kit

For cell cycle studies, unsynchronized T47D cells were seeded at 250,000 cells/well in RPMI (Biowest, Nuaillé, France) 10% FBS medium supplemented with L-glutamine (2 mM), HEPES (10 mM), sodium pyruvate (1 mM) and PEST. Cells were treated with VEH (0.05% DMSO), 4-OHTAM (5 µM) or test compounds (5 µM) for 24 to 72 h. Cells were then washed in ice-cold PBS and fixed in cold 70% ethanol at −20 °C overnight. Then, the cell pellet was resuspended in FACs buffer (2 mM EDTA, 2% FBS in PBS) and incubated with Ki67-APC antibody (Miltenyi Biotec, Auburn, CA, USA) and DAPI (4′,6-diamidino-2-phenylindole; Sigma-Aldrich) for 20 min at room temperature. Finally, cells were washed and resuspended with FACs Buffer for cell cycle analyses. For apoptosis studies, an Annexin V-FITC Apoptosis Kit (Miltenyi Biotec) was used. Briefly, treated cells were collected in 100 μL of ice-cold AnnexinV binding buffer (10 mM HEPES pH 7.4, 140 mM NaCl, 2.5 mM CaCl₂) containing 10 μL of Annexin-V FITC and incubated for 15 min at room temperature. After that, cells were washed with 1 mL binding buffer, sedimented at 300× g 10 min, and resuspended with 500 μL binding buffer containing 5 μL of propidium iodide (PI; 100 μg/mL) prior to acquisition in MACSQuant10 cytometer (Miltenyi Biotec) where 10,000 or 20,000 events were used for cell cycle and apoptosis analysis, respectively.