## 3 Results
### Chemical Comparison and Mock Treatment Are Inaccurate Markers of Cellular Responses
The concern with using the high nspDBD cell culture concentrations was that it would reduce Jurkat viability to zero, leading to results that would be difficult to detect and would not highlight responses due to small changes in concentration. The measured nspDBD DI water concentrations were comparable to the concentrations known to induce cellular responses in Jurkat. Therefore, these concentrations were added to cells in culture medium and incubated for 24 h. This mock treatment would not be comparable to the plasma treatment directly but rather an indication of how Jurkat cells reacted to the oxidative environment. The viability and oxidative stress responses of Jurkat cells are shown in Figure 6, with the results obtained after plasma treatment as a benchmark. Mock treatment at the COST-Jet conditions did not reduce cell viability to the degree caused by plasma treatment (Figure 6a,d), showing that H\({}_{2}\)O\({}_{2}\) concentrations below 20 uM did not adversely affect the cells. The H\({}_{2}\)O\({}_{2}\) concentrations greater than 100 uM induced higher magnitudes of cell death than the nspDBD conditions (Figure 6a,d). These results supported previous literature that demonstrated H\({}_{2}\)O\({}_{2}\) concentrations above 100 uM induced \begin{table}
\begin{tabular}{c c c} \hline \hline
**Treatment Condition** & **H\({}_{2}\)O\({}_{2}\) (uM)** & **NO\({}_{2}\)\({}^{-}\) (uM)** \\ \hline COST-Jet: 2 min & 6.50 & 1.15 \\ COST-Jet: 4 min & 17.35 & 2.01 \\ nspDBD: 45 Hz & 108.78 & 3.92 \\ nspDBD: 75 Hz & 149.93 & 9.01 \\ \hline \hline \end{tabular}
\end{table} 
Table 2: Mock Treatment Concentrations. cellular arrest in Jurkat cells [41]. This clearly showed, as described above, that the higher oxidative environment due to the high concentration of H\({}_{2}\)O\({}_{2}\) adversely affected the Jurkat cells. Added NO\({}_{2}\)\({}^{-}\) at concentrations measured from the nspDBD and COST-Jet treatments did not have any effects on cell viability. In addition, when NO\({}_{2}\)\({}^{-}\) was combined with H\({}_{2}\)O\({}_{2}\), the observed loss in viability was comparable to cells treated with H\({}_{2}\)O\({}_{2}\) alone. These observations suggested that NO\({}_{2}\)\({}^{-}\) did not influence the cell viability directly or through reaction with H\({}_{2}\)O\({}_{2}\). Mitochondrial activity was stimulated by H\({}_{2}\)O\({}_{2}\) alone and in combination with NO\({}_{2}\)\({}^{-}\) at the nspDBD conditions in comparison to the untreated control, as shown in Figure 6b,e. H\({}_{2}\)O\({}_{2}\) is a potent oxidizer and is well known to induce mitochondrial stress at concentrations as low as 100 \(\upmu\)M [50]. NO\({}_{2}\)\({}^{-}\) alone had no discernable effect on mitochondrial O\({}_{2}\)\({}^{-}\) production for either of the mock treatments. The significant increase in mitochondrial activity due to the addition of exogenous H\({}_{2}\)O\({}_{2}\) in comparison with the plasma-treated cells was indicative of the strong oxidative environment. The H\({}_{2}\)O\({}_{2}\) concentration produced by the COST-Jet for both conditions was too low to stimulate mitochondrial O\({}_{2}\)\({}^{-}\) above the untreated control. The effect of mock treatment on ecto-CRT expression was evaluated on Jurkat cells incubated with the RONS for 24 h. As seen in Figure 6c,f, only the nspDBD concentration of H\({}_{2}\)O\({}_{2}\) produced a slight rightward shift and a second brighter peak in fluorescence. This was due to the higher numbers of dead and dying cells, as seen in Figure 6a,d. None of the other mock treatments produced an increase in ecto-CRT expression, clearly indicating that stable chemistry is unable to trigger necessary biological pathways that cause CRT translocation. Figure 6: Exogenous addition of H\({}_{2}\)O\({}_{2}\) elicited cellular responses from Jurkat T lymphocytes at the two comparable conditions of 2 min/45 Hz (**a**–**c**) and 4 min/75 Hz (**d**–**f**).