## 3 Results
### Experimental Setup III: Identical Cell Number Seeded in Different Media  
The effects of CAP on the cells are mediated by the plasma-activated medium. To address the question to what extent the choice of medium influences these effects, we cultured OMYleb and OMYsd cells in DMEM medium or L-15 medium (Figure 5). L-15 medium has been used for decades for culturing cells of salmonid origin [41; 42]. The composition of the two media used is listed in Table S2.  
Figure 4: The influence of plasma treatment (PAM) on cell viability at different cell numbers. Fish and mammalian cells were seeded in different numbers (\(2\times 10^{4}\) to \(6\times 10^{4}\)) to reach the same degree of confluency (\(\sim\)80%) at the time of stimulation. The increase of cell number had a strong effect on RTgill-W1, but not on OMYsd cells. Cells were grown under identical medium conditions (\(n=3\)).  
Figure 5: Cell viability after plasma treatment (PAM) when identical numbers of cells were seeded initially. Cells are grown under different medium conditions (DMEM vs. L-15 medium). Note the protective effect of L-15 against the strong PAM120 disturbance (\(n=3\)).  
The cultivation in L-15 medium previously treated with plasma for 15 s, 30 s and 60 s resulted in a decrease of viability, while cells grown in DMEM--treated for 15 s, 30 s and 60 s--were hardly affected. However, treatment with PAM120 generated from DMEM drastically reduced the viability of OMYleb (\(-80.4\%\) compared with the untreated control) and OMYsd (\(-87.4\%\)). In contrast, the same cells in PAM120 generated from L-15 medium were reduced in their viability by only \(-52.8\%\) and \(-34.9\%\), respectively. Thus, L-15 medium had a protective effect against CAP for both fish cell lines.  
Since the effect of the plasma is mediated via the liquid, the varying compositions of the different media used may influence the effectiveness of the PAM. The comparison of the antioxidant capacity of the two media (Figure 6) revealed that the L-15 medium (containing 5 mM pyruvate) has a higher oxidative capacity than DMEM medium (which originally contains 1 mM pyruvate). To determine if the oxidative capacity of DMEM depends on the pyruvate content, we added different pyruvate concentrations to DMEM whose pyruvate content was adjusted to that of the L-15 medium and then re-measured the oxidative capacity of these media. We found that DMEM without or with 1 mM pyruvate shares a similar degree of oxidative capacity. After supplementation of 5 mM pyruvate, the antioxidative capacity of DMEM increased enormously (+110%). The capacity of the DMEM medium with 5 mM pyruvate exceeded that of the L-15 medium, which also contains 5 mM pyruvate.  
In addition, we cultured the two mammalian and three picse cell lines in DMEM without pyruvate. Omitting the pyruvate from the medium did not alter the general viability of the cells (data not shown). However, the viability was in part significantly impaired when the cells were stimulated with plasma-treated pyruvate-free medium (Figure 7). A treatment time of only 60 s of pyruvate-free DMEM significantly reduced the viability of OMYleb and OMYsd cells compared with 60-s treatment of DMEM containing pyruvate. By contrast, the measured viability of the other cells (RTgill-W1, mHep-R1 and HepG2) was unchanged in pyruvate-free PAM compared with PAM containing pyruvate.  
Figure 6: Antioxidant capacity of DMEM (containing 0 mM, 1 mM or 5 mM pyruvate) and L-15 medium (containing 5 mM pyruvate). The values of DMEM 0 mM were set as 100% (\(n=4\)).