The electric field effect on thefluorescence lifetime of NADHmay be evidence of the occurrence of apoptosis and/or necrosis. Ina previous study, no major changes were observed in the autofluorescenceof NADH under H2O2-induced necrosis in boththe HeLa and 143B cells.27 On the otherhand, an increase in the NADH autofluorescence lifetime was observedduring staurosporine (STS)-induced and N-methyl-N-nitro-N-nitrosoguanidine-induced apoptosisin the HeLa cells.27,28 Then, we examined the relationshipbetween the autofluorescence lifetime of NADH and apoptosis in bothWFB and W31 cells using a 1 μM concentration of STS. The inductionof STS is known to release cytochrome c from mitochondriaand activate caspase-3,29 which is oneof the caspase groups that are executioners of apoptosis. It was foundthat the autofluorescence lifetime of NADH increased after the additionof 1 μM STS in both normal and cancer cells (Figure S4). Therefore, it is concluded that application ofnsPEF induces apoptosis both in normal (WFB) and cancer (W31) cells,as in the case of the HeLa cells.24,25 Apoptosisis traditionally defined by morphological criteria, such as cell shrinkageand cell surface blebbing. In fact, some morphological changes weredetected after application of nsPEF in our study in both the celltypes, depending on the amplitude and pulse width of the applied electricfield, as shown in Figures S5 and S6. Thedetection of apoptosis using the fluorescence lifetime of NADH hasa great advantage of confirming apoptosis with label-free detection,and this method has been applied in our previous study regarding thenanosecond pulsed field-induced apoptosis of HeLa cells25 and also in a recent study by another group.30
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