2.5. Annexin V Assay and Caspase 3/7 (Cas 3/7) Activity

Cells (Ca9-22, CAL 27, and SG) were stained with an annexin V-FITC (1:1000 dilution) (Strong Biotech Corporation, Taipei, Taiwan)/7AAD (1 μg/mL) solution at 37 °C for 30 min [42]. After PBS resuspension, cells were analyzed by a flow cytometer. Cas 3/7 activation is associated with apoptosis signaling, which was detected using a Glo^® 3/7 commercial system (Promega; Madison, WI, USA) [16]. A Cas 3/7 substrate (DEVD) was added to the medium at 37 °C for 30 min. Consequently, apoptotic cells containing activated Cas 3/7 could cleave DEVD to generate the luminescent signal detected by the luminometer.