Apoptosis Assessment by Annexin V-7-AAD

Many agents, including the clinically useful sorafenib, can induce apoptosis [20 (link)]. Following 24-h treatment with 8 μM sorafenib, PANC-1–derived clones were trypsinized, washed with cold PBS, and resuspended in PBS. The advantage of 7-amino-actinomycin D (7-AAD) over propidium iodide is that there is minimal spectral overlap between the emissions. We used a fluorescein isothiocyanate (FITC) Annexin V Apoptosis Detection Kit with 7-AAD (#640922; BioLegend, USA) according to the manufacturer’s instructions. Briefly, 5 μL FITC–annexin V and 5 μL 7-AAD Viability Staining Solution (#00-6993; eBioscience, USA) were added to a 100-μL cell suspension (1 × 10⁶ cells) in binding buffer. The cells were gently vortexed and incubated for 15 min at room temperature in the dark, and 400 μL binding buffer was added for flow cytometric analysis using a FACScan Flow Cytometer (Becton Dickinson).