Annexin 5 fluorescein isothiocyanate fitc labeled apoptosis detection kit 1

An annexin V–fluorescein isothiocyanate (FITC)-labeled Apoptosis Detection Kit I (Pharmingen, San Diego, CA, USA) was used to detect and quantify apoptosis by flow cytometry according to the manufacturer’s instructions. In brief, MCF-7:5C cells (1 × 10⁶ cells/mL) were seeded in 100-mm dishes and cultured overnight in estrogen-free RPMI 1640 medium containing 10% SFS. The next day, cells were treated with <0.1% ethanol vehicle (control) and E₂ (1 nM) for 96 hours and then harvested in cold PBS (Invitrogen) and collected by centrifugation for 10 minutes at 500 × g. Cells were then resuspended at a density of 1 × 10⁶ cells/mL in 1 × binding buffer (HEPES buffer, 10 mM, pH 7.4, 150 mM NaCl, 5 mM KCl, 1 mM MgCl2 and 1.8 mM CaCl2) and stained simultaneously with FITC-labeled annexin V (25 ng/mL; green fluorescence) and propidium iodide (PI) (50 ng/mL). PI was provided as a 50 μg/mL stock (Pharmingen) and was used as a cell viability marker. Cells were analyzed using the BD LSR II flow cytometer (BD Bioscience, San Jose, CA, USA), and the data were analyzed with CellQuest software.

GH3 cells were treated with vehicle control or RE, or RE in the presence or absence of pretreatment with 100 μM Z-VAD-fmk, a pan-inhibitor of caspase for 1 h. Following treatment, apoptosis was assessed using the Annexin V-fluorescein isothiocyanate (FITC)-labeled apoptosis detection kit I (BD Biosciences Pharmingen, San Diego, CA, USA). Cells were washed twice with phosphate-buffered saline (PBS), suspended in binding buffer and stained with Annexin V-FITC and propidium iodide. Cells undergoing apoptosis were detected by flow cytometry FACS Calibur (Becton Dickinson, Rutherford, NJ, USA).