Quantification of Apoptosis and Necrosis

A549 cells (1 × 10⁴ cells/well) were incubated in each well of a 24-well plate with the most potent compounds in this series at IC₅₀ concentrations for 15 h. Then, an apoptotic/necrotic cell detection kit (PromoKine, Heidelberg, Germany) was used according to manufacturer′s instructions with some modifications [43 (link),44 (link)]. Briefly, the cells were washed twice with 1× binding buffer, a staining solution containing 50 μL of 1× binding buffer, 5 μL of FITC-Annexin V solution, and 5 μL of ethidium homodimer III solution, and treated for 30 min at room temperature in a protected-light environment. After washing of cells in 1× binding buffer, cells were analyzed under a Biorevo BZ-9000 all-in-one fluorescence microscope (Keyence, Osaka, Japan). The number of apoptotic cells, late apoptotic or necrotic cells, and necrotic cells was quantified as previously described [45 (link)].