Fitc annexin 5 apoptosis detection kit

HepG₂ cells were treated with and without ATO according to the aforementioned concentrations for 24 hours. Following treatment, the cells were isolated by trypsinization and rinsed twice with ice-cold PBS. Detection of phosphatidylserine on the outer leaflet of apoptotic cells was performed using Annexin V-FITC Apoptosis Detection kit in combination with propidium iodide (PI) staining (BD Pharmigen, Indianapolis, IN) according to the manufacturer’s recommendations [49 (link),50 (link)]. The samples were pelleted and resuspended in binding buffer at 1 × 10⁶ cells/mL. Tubes were labeled for each sample with 5 μL of Annexin V FITC, 5 μL of dissolved PI and 100 μL of the cell suspension (100,000 cells) being added to each tube. Each sample was examined by flow cytometry using a FAC Sort (Becton Dickinson Co, San Diego, CA, USA). The percentages of apoptotic cells were determined by analysis of the dot plots using Cell Quest software.

MIA PaCa-2, PANC-1, and BxPC-3 cells (1–2×10⁵)/well were seeded in 6 well plates, allowed to attach overnight, and then received indicated treatments for 48 hrs. Cells were washed with cold PBS, then resuspended and stained with Propidium iodide (PI) and Annexin V-FITC using Annexin V–FITC Apoptosis Detection Kit (BD Pharmigen, 556547) according to the manufacturer’s protocol. The resulting fluorescence was measured by a flow cytometer (Bio-Rad ZE5 Analyzer).