## 3 Results and Discussion
### Analysis of Cell Death after Microplasma Treatment  
A flow cytometry analysis was conducted on set C samples (Figure 1) in order to examine the potential relationship between lipid alterations and cell death. Cell death can occur in two forms: accidental cell death (i.e., necrosis) and regulated cell death (e.g., apoptosis, ferrotptosis, autophagy, etc.) [36]. We employed an Annexin V binding assay, which enabled the discrimination of live cells, early apoptotic cells, late apoptotic cells, and necrotic cells in both control and microplasma-irradiated cells.  
Annexin V is a calcium-dependent protein that specifically binds PS on the outer leaflet of the cell membrane. In healthy cells, PS is normally found in the inner leaflet (cytoplasmic site) of the cell membranes. However, during apoptosis, PS translocates to the outer leaflet of the cell membrane. This translocation of PS is detected through FITC conjugated Annexin V. Propidium iodide is often used along with Annexin V for studying apoptosis. It selectively labels nucleic acids of dead and late apoptotic cells. Single positive for Annexin V staining indicates apoptotic cells, while double-positive staining for Annexin V and propidium iodide marks necrotic cells [37].  
Our results revealed that microplasma irradiation at 4.0 kV without further incubation (designated as C4K0H cells) led to a significant increase in live cells (\(p\) = 0.002), accompanied by a significant decrease in apoptotic (\(p\) = 0.005) and necrotic cells (\(p\) = 0.0002) compared to the control samples (Figure 6). The notable cell death observed in the control samples was likely due to the inherent factors of the staining procedure, which included trypsinization, washing, centrifugation, and exposure to staining reagents at room temperature. Thus, microplasma irradiation appears to exert a protective effect against apoptosis and necrosis in C4K0H cells, potentially mediated by short-lived species generated in the aqueous medium by the microplasma [38].  
On the other hand, microplasma irradiation at 4.0 kV followed by 12 h incubation (C4K12H cells) did not exhibit significant differences in live cells and apoptotic cells compared to the controls. However, early apoptosis was significantly (\(p\) = 0.0006) increased, while necrosis was significantly (\(p\) = 0.0002) decreased in C4K12H cells relative to the controls. These findings suggest that the effects of microplasma irradiation no longer exist 12 h after the treatment.  
Furthermore, irradiation at 4.5 kV with no subsequent incubation (C4.5K0H cells) significantly increased live cells (\(p\) = 0.004) and reduced apoptotic (\(p\) = 0.009) and necrotic cells (\(p\) = 0.0002). Nonetheless, compared to C4K0H cells, live cells in C4.5K0H were diminished, while early and late apoptotic cells, as well as necrotic cells, were approximately twofold higher. These data support that the 4.0 kV condition is more favorable for cell viability than the 4.5 kV condition.  
It is worth noting that cell death by irradiation is not limited to apoptosis and necrosis. Cells may undergo other forms of regulated cell death, including ferprotosis [39; 40], which was not explored in this study.  
Figure 5: Changes in lipids within 10 min following microplasma irradiation at 4.5 kV condition. (**a**) Changes in total cell membrane-associated lipids, and (**b**) total non-cell membrane lipids were calculated based on the absolute area values obtained from LC/MS analysis (set #2 data). Statistical analysis was performed to compare the B4.5K0H group (n = 5) with the control group (n = 5). (**b**) Relative changes (expressed in percentages) in (**c**) glycerophospholipids and (**d**) sphingolipids in the B4.5K0H group were compared to those in the control group.