Assessment of Apoptosis by Annexin V-PE/7AAD

BioLegend's PE Annexin V Apoptosis Detection Kit with 7AAD (Biolegend, USA) was used to assess the apoptotic effect of 1a compound as previously described.^{[6e (link), 18a ]} Briefly, 0.5 x 10⁵ cells per 1 ml of complete culture medium were seeded in a 24-well culture plate, treated with 1a compound in different concentrations (2.5, 5 and 10 μM) for 72 h. An untreated sample was also included as a negative control. Treated and untreated cells were then harvested and washed twice with cold BioLegend's Cell Staining Buffer, transferred to the polystyrene round-bottom tubes (BD Bioscience, USA) and stained with 2 μl of PE-conjugated Annexin V and 2 μl of 7-AAD solution for 15 min at room temperature in the dark. 300 μl of Binding Buffer was added to each tube and analyzed immediately by four-color FACSCalibur flow cytometer (BD Bioscience, USA) with proper setting. The data were analyzed by FlowJo software packages.