## 3 Results
### Measurements of Cellular Responses to Synergistic Treatment of CAP with H2O2  
To find the cause of the synergistic bactericidal effects, some important factors related to cytotoxicity were measured. The pH of the solution was measured after CAP treatment (Figure 4a). pH is a very important factor in determining bacterial membrane potential and disinfection efficiency [17]. Since H2O2 is acidic, the pH of 1% H2O2 solution was initially slightly lower than that of DI water. It was dramatically reduced with CAP treatment, and more reduction was observed with combination treatment. However, the pH values were not significantly different between CAP only treatment and CAP with H2O2 treatment. Figure 3: Bactericidal effects of CAP on the bacterial suspension compared with H2O2; (**a**) A graph showing CFU decrease according to plasma treatment time; (**b**) A graph showing CFU decrease according to H2O2 concentrations with or without CAP treatment; (**c**) SEM images of the bacteria treated with CAP, H2O2 and CAP together with H2O2. Scale bars are 400 nm. Lipid peroxidation was generally accepted as the first process of plasma-cell interactions. To examine whether membrane damage was the reason for the synergistic effects, the amount of MDA was measured. MDA is formed by the degradation of polyunsaturated lipids and is therefore used as a marker of oxidative stress. Figure 4b shows the increase in MDA concentration with CAP treatment. However, the differences between the 1% H\({}_{2}\)O\({}_{2}\) and DI solutions were not significant. While H\({}_{2}\)O\({}_{2}\) shows slightly higher values in MDA quantification without CAP treatment, the lipid oxidation was somewhat reduced with combined treatment of CAP and H\({}_{2}\)O\({}_{2}\). Similarly, Figure 4c shows that the intracellular ROS level increased with CAP treatment. These results imply that CAP induced oxidative stress inside a cell. However, MFI of H2 DCFDA were decreased in the 1% H\({}_{2}\)O\({}_{2}\) solution, which would be related to cellular defense process to H\({}_{2}\)O\({}_{2}\). Figure 4d shows the MFI of Flipr dye per a cell, which represents the trans-membrane potential. The Flipr fluorescence is known to become brighter when the membrane is electrically de-polarized. The intensity was not changed much with CAP treatment but increased with H\({}_{2}\)O\({}_{2}\) treatment. More enhancement of MFI was observed with the combination treatment. In contrast to MDA measurement, trans-membrane potential was affected more significantly by H\({}_{2}\)O\({}_{2}\) than CAP, and the change became more significant in a combination treatment. This result suggests the differential influence of H\({}_{2}\)O\({}_{2}\) and CAP on the cell membrane. H\({}_{2}\)O\({}_{2}\) may pass through the membrane easily than other species from CAP, or H\({}_{2}\)O\({}_{2}\) can freeze some proteins working for maintaining transmembrane potential. Figure 4e shows that the membrane permeability was similarly increased as membrane potential depolarization. The intensity of membrane impermeable PI dye increased in 1% H\({}_{2}\)O\({}_{2}\) and increased more when treated simultaneously with 1% H\({}_{2}\)O\({}_{2}\) and CAP. Figure 4: Measurements of factors related to bactericidal effects in the combination of CAP and H\({}_{2}\)O\({}_{2}\); (**a**) pH; (**b**) MDA concentration; (**c**) Mean values of the intensity of H2 DCFDA dye relative to the control; (**d**) Mean values of the intensity of Flipr dye relative to the control; (**e**) Mean values of the intensity of PI dye per a cell relative to the control; (**f**) Mean values of the intensity of 8-OH-dG dye relative to the control. The notations ‘+’ and ‘\(-\)’ mean the condition with and without each treatment, respectively. As a following intracellular oxidation signal, we used antibody to deoxyguanosine (8-OHdG), which is one of the major products of DNA oxidation. DNA damage is a very important signal in cells and is generally accepted as a key process of cell death.