FACS Analysis of Apoptosis and Cell Cycle

The fluorescence-activated cell sorting (FACS) technique was employed to analyze the 3c- and 3e-induced cell death, the activation of executive caspase-3, and the cell cycle in the A549 cells. The quantitative analysis of apoptosis and necrosis was performed using an Annexin V-fluorescein isothiocyanate (FITC)/ propidium iodide (PI) apoptosis kit (BD Biosciences, BD Pharmingen™, San Jose, CA, USA) as previously described [36 (link)]. To determine the active form of caspase-3, the A549 cells were plated into 6-well plates at a density of 6 × 10⁵ cells/well. The next day, the growth medium was replaced with a fresh one supplemented with compound 3c or 3e (10, 20, and 40 µM) or the DMSO vehicle (0.1%; control cells). After 48-h incubation, the samples were harvested and the level of active caspase-3 was determined using the phycoerythrin (PE) Active Caspase-3 Apoptosis Kit (BD Pharmingen™) according to the manufacturer’s instructions. The stained cells were analyzed using FACS Calibur, and data were analyzed using Cell Quest Pro Version 6.0 (BD Biosciences, San Jose, CA, USA) for the Macintosh operating system. The results were calculated as a percent of cells with active caspase-3 among all the analyzed cells. The cell cycle analysis was performed by determination of the DNA contents on the basis of PI staining as previously described [36 (link)].