2.13. Annexin V staining

Apoptosis analysis was performed using PE Annexin V Apoptosis Detection Kit I (BD Pharmingen™, San Jose, CA, USA) following the manufactory's protocol. 2.5 × 10⁵ cells were seeded in duplicate in 6-well plates and on the next day, the cells were transfected with 60 nM Control and APE1 siRNA. On the following evening, the cells were split again into 12 well plates (1.5 × 10⁵ cells/well) as triplicates. 48 h after transfection, cells were treated with ABS (100 μM, pH 4.0) or PBS for 20 min, followed by recovery in complete media for 3 h. Cells were then harvested and stained with Annexin-V and propidium iodide (PI). The cells were washed with PBS and re-suspended in a binding buffer (HEPES buffered saline solution supplemented with 2.5 mM CaCl2) and then subjected to fluorescence-activated cell sorting (FACS) analysis using a flow cytometer (Becton Dickinson). Apoptotic cell death was determined by counting the cells that stained positive for Annexin-V.