## 4 Discussion
### Fish Cells Respond Differently to Plasma Treatment than Mammalian Cells
Thus, our study provides evidence that the observed effects are produced by other reactive species (NO\({}_{2}\)\({}^{-}\), NO\({}_{3}\)\({}^{-}\) and others). [PERSON] et al. investigated the effect of CAP on human keratinocytes cultivated in IMDM medium (Iscove's modified Dulbecco's medium), which is a strong radical scavenger, and in RPMI medium, which is a weak radical scavenger. The viability of cells in IMDM decreased to 50% after 30 s of CAP, while the number of cells in RPMI reduced to 15% after identical treatment [27]. These studies are further evidence that the effect of CAP is mainly mediated by oxygen radicals and their longer-lasting reaction products. In general, the L-15 medium, in contrast to DMEM, is based on a CO\({}_{2}\)-independent buffer system and therefore contains higher concentrations of phosphates and various amino acids but lacks sodium bicarbonate (Table S2). In addition, the L-15 medium contains 5 mM pyruvate, whereas the DMEM medium used here contains 1 mM pyruvate only. It has been shown that high concentrations of pyruvate can have a protective effect on human lung cancer and osteosarcoma cells or murine liver cells by reacting with reactive oxygen species (ROS), which in turn reduces the pyruvate concentration [33; 55; 56]. Measurements of the hydrogen peroxide concentration revealed low concentrations of hydrogen peroxide after plasma treatment of a low-pyruvate medium and storage of the medium for 24 h [33]. However, when using a medium with 5 mM pyruvate (either DMEM or L-15), we could not detect hydrogen peroxide even with prolonged treatment times of 240 s (see Supplementary Materials Figure S2). Therefore, in our view, it is not sufficient to measure the hydrogen peroxide concentration in the media, at least not for media with a high pyruvate content. For the exact comparison, we suggest measuring the total antioxidant potential. We analysed the antioxidative potential of DMEM and L-15 medium using a commercial assay that quantifies the overall amount of antioxidative species in the solution. The combined action of all the antioxidants present in a given medium provide synergistic protection against reactive oxygen or nitrogen species. Therefore, we determined the total antioxidant capacity of each medium instead of adding up the antioxidant capacities of the individual components. Special attention was paid to pyruvate, as former experiments pointed to its role in neutralizing CAP-generated reactive oxygen species [33]. We did prove, however, that the overall antioxidative capacity of a medium is not only dependent on the pyruvate content. The antioxidative capacity of the DMEM medium was higher than in the L-15, although both media contained 5 mM pyruvate. To take the role of pyruvate into account, we also varied this parameter in our experiments and carried out cell culture experiments with PAM generated from a DMEM medium, which lacked pyruvate. After assessing the general vitality of the cells in a pyruvate-free medium, we carried out the plasma treatment, which reduced the viability of two of the three trout cell lines (OMYleb and OMYsd cells). This effect was not observed in RTgill-W1, mHep-R1 and HepG2 cells. This suggests that the cellular responses can vary greatly depending on the cell type. Some cell types (OMYleb, OMYsd) react more sensitively to the loss of an antioxidant agent in PAM, while other cells are able to compensate for this, at least over the relatively short period of investigation (24 h). In comparative experiments on the action of CAP or PAM, it is therefore mandatory to ensure a congruent media composition, as even small deviations (e.g., in pyruvate content) can lead to an altered cell sensitivity. Other potentially antioxidant substances may be included in the cell culture media used. These include above all phenol red, amino acids [57; 58], proteins, carbohydrates [59] and vitamins such as pantothenic acid (VitB5), riboflavin (VitB2) and folic acid [60; 61; 62]. RONS formed in the medium by the action of CAP can react with these media components.