## 4 Discussion
However, this does not mean that other used media are less cytotoxic since hydrogen peroxide could have been converted into hydroxyl radicals or resulted in the peroxidation of organic molecules. Moreover, reactive species other than hydrogen peroxide and nitrites are generated at the plasma-liquid interface. When we compare different cell lines with the same culture medium requirement, striking differences in metabolic impairment emerge as shown in Table 3. This observation implies that the intrinsic properties of tumor cells are the main factor determining the response to plasma treatment. Since different cell lines have different growth rates based on different cell culture medium requirements, optimal cell concentrations, and characteristic doubling times, the metabolic activity and protein content of each cell line were always normalized to the non-treated control of the same cell line, thereby eliminating possibly confounding factors related with experimental design. This procedure helped us to establish dose-response curves and calculate the time required to inhibit the metabolic rate and protein content by 50% (IT50), analogous to IC\({}_{50}\) (half maximal inhibitory concentration). In order to allow a better comparison between different cell lines, we plotted the calculated IT50 and associated 95% confidence interval of each cell line on a graph and the calculated average IT50 as shown in Figure 5. With this reference line, we assigned lower than average IT50 to a positive response and higher than average IT50 to a negative response. We attempted to categorize response to plasma treatment according to metabolic rate and proliferation rate. By doing so, we classify three different types of answer: (1) resistant, (2) susceptible, (3) stressed, and (4) stunned. When first studying the effect of plasma in metabolic activity by MTT assay, HFF1, WIDR, OE19, HCC1806, and MCF7 cells showed the highest IT50, meaning that these cells were not entirely susceptible to the plasma's effect. Indeed, HFF1, WIDR, OE19, and HCC1806 cells remained viable until longer treatment durations compared to the other cell lines as seen in SRB assays. These cells were considered resistant to plasma. However, that was not the case for MCF7 cells. The viability of these cells promptly decreased with brief treatments, and was, paradoxically, the cell line with the lowest treatment duration required to decrease protein content by 50% as seen by SRB assay. These cells were considered to be stressed. Another group of cell lines, PC3 and MNNG/HOS, reported a decrease in metabolic activity but not in protein content and were considered stunned. Finally, susceptible cells showed the lowest IT50 for metabolic activity with concordant lowest IT50 for proliferation rate. In order to understand this classification, two concepts were introduced. Firstly, the amount of SRB concentration is directly proportional to the number of living cells [72] and, therefore, can be extrapolated to measure cell proliferations [73], since it is not likely that SRB will overestimate cell number due to cell debris [74]. Secondly, the MTT assay is based on the capacity of metabolically active cells to catalyze the reduction of tetrazolium salt. This reaction occurs mainly in mitochondria and depends on membrane permeability, mitochondrial mass, and activity [75]. Therefore, the SRB assay measures proliferation, and MTT measures metabolism. Alterations in proliferation and metabolism are usually concordant. Proliferating cells tend to have higher metabolic rates, parallel to biosynthesis, when matched to those of non-dividing cells. Similarly, a reduction in proliferative rate is typically accompanied by a reduction in metabolic rate, as seen in susceptible cells. The reduction in proliferative rate can be caused by cell death or cycle arrest. Many authors report that low doses of plasma preferably induce cell cycle arrest, and higher doses of plasma lead to apoptosis and necrosis in cancer cells [76].