## 3 Results
### Chemical Comparison and Mock Treatment Are Inaccurate Markers of Cellular Responses  
The comparable cellular responses from COST-Jet and nspDBD treatments suggested that, if the cellular stress response mechanisms were the same, the stressors generated by the plasma should be of similar magnitude. These responses have been attributed to multiple factors from plasma treatment, e.g., pulsed electric fields [39], RONS [40]. While studies have shown that CRT translocation and mitochondrial O\({}_{2}\)- production is stimulated by the presence of short-lived species [15], Jurkat  
Figure 4: Expression of calreticulin (ecto-CRT) was measured 24 h post-treatment on Jurkat T lymphocytes treated at the two comparable conditions: 2 min/45 Hz (**Left**) and 4 min/75 Hz (**Right**). Both devices induced similar increases in the numbers of ecto-CRT-positive cells. Figure 3: Mitochondrial O\({}_{2}\)– production in Jurkat T lymphocytes in response to plasma treatment. COST-Jet (2 and 4 min) and nspDBD (45 and 75 Hz) induced higher and comparable magnitudes of oxidative stress relative to the untreated control cells. This experiment was repeated once in triplicate. The data plotted represent the mean and standard deviation. T-lymphocytes are known to be sensitive to a few hundred micromolar of H\({}_{2}\)O\({}_{2}\)[41], a long-lived species. To investigate the contribution of long-lived species to the biological responses observed by plasma treatment, we measured their concentration subsequent to plasma treatment and subjected the cells to these measured concentrations as a mock chemical treatment. We hypothesized that similar concentrations of H\({}_{2}\)O\({}_{2}\) and NO\({}_{2}\)\({}^{-}\) must be produced to achieve similar biological responses. We evaluated their concentrations at both conditions (2 min vs. 45 Hz; 4 min vs. 75 Hz) that caused similar reductions in viability in order to correlate the increase in solvated RONS with this biological endpoint. While nitrate (NO\({}_{3}\)\({}^{-}\)) is an important long-lived species to measure for characterization of liquid chemistry, it was previously shown to have an insignificant impact on cellular viability in multiple cell lines when mixed with H\({}_{2}\)O\({}_{2}\) and NO\({}_{2}\)\({}^{-}\)[42]. Therefore, we focused our experiments on H\({}_{2}\)O\({}_{2}\) and NO\({}_{2}\)\({}^{-}\). First, we measured the long-lived species in the cell culture medium to understand the concentrations to which the cells were exposed after plasma treatment. Colorimetric assays were unaffected by time-dependent losses in reactive species since the assay reaction times (10 min for H\({}_{2}\)O\({}_{2}\) and 30 min for NO\({}_{2}\)\({}^{-}\)) were considerably shorter than the amount of time required for significant H\({}_{2}\)O\({}_{2}\) and NO\({}_{2}\)\({}^{-}\) losses due to reactivity [24]. Both long-lived species were produced at higher concentrations from nspDBD treatments when compared with the COST-Jet-treated solutions (Figure 5). The concentration of H\({}_{2}\)O\({}_{2}\) (Figure 5, left) and NO\({}_{2}\)\({}^{-}\) (Figure 5, right) increased with longer treatment times for the COST-Jet and higher frequencies for the nspDBD. In culture medium treated by the COST-Jet, an increase in exposure time resulted in increased concentrations of H\({}_{2}\)O\({}_{2}\) (78.7 and 103.6 \(\upmu\)M after 2 and 4 min, respectively) and NO\({}_{2}\)\({}^{-}\) (3.6 and 7.1 \(\upmu\)M, respectively). The nspDBD concentrations increased with frequency for both species: 187.3 \(\upmu\)M (45 Hz) to 226.9 \(\upmu\)M (75 Hz) for H\({}_{2}\)O\({}_{2}\) and 27.8 \(\upmu\)M (45 Hz) to 47.9 \(\upmu\)M (75 Hz) for NO\({}_{2}\)\({}^{-}\). These concentrations in the medium were much higher than those reported previously in deionized water [11; 14], which was inconsistent with the understanding that cell culture medium would buffer long-lived species production in the liquid due to reactions between plasma-generated short-lived predecessors (such as OH and NO) and organic compounds.