## 3 Results
### Activation of Immune Cells with Plasma Treatment  
Cellular activation can be assessed using several methods such as calcium signaling. Fluo-3 is a labelled calcium indicator, and its fluorescence increases 100-fold upon calcium binding. Calcium influxes were observed in early lymphocyte activation. Total cellular fluorescence was measured by flow cytometry in macrophages (Figure 3a) and T cells (Figure 3b). After addition of Fluo-3 labeled splenocytes to plasma-treated melanoma cells, there was no significant change in calcium influx in macrophages (Figure 3c) and T cells (Figure 3d). Of note, a significant calcium influx was demonstrated for splenocytic T cells directly exposed to plasma and over time (Figure 3c) but not in macrophages (Figure 3d). Calcium release and protein kinase activity are closely linked. Being a member of the Ras-Raf-Erk signal transduction cascade, the Erk1/2 (extracellular signal-regulated Kinase-1/2) kinases are activated via phosphorylation. Upon activation, the Erk kinases function in cellular proliferation, differentiation, and survival. A significant increase (Figure 3e) of phosphorylated Erk1/2 was demonstrated for plasma-treated T cells after 1 h and 4 h (Figure 3f). For splenocytes  
Figure 2: Treatment of murine immune cells with physical plasma. (**a**) Representative murine spleen after harvesting and prior to homogenization; (**b**) metabolic activity of naïve (-PMA) and activated (+PMA) splenocytes after plasma treatment; (**c**) cytokine/chemokine profile 24 h after 120 s plasma treatment in naïve splenocytes. Data are mean +S.E. of several spleens, statistical analysis was performed using t test. PMA: phorbol 12-myristate 13-acetate. co-cultured with plasma-treated B16F10 melanoma cells, a small but significant increase was observable 1 h after culture onset (Figure 3f). This suggests that plasma treatment and plasma-treated melanoma cells may be able to stimulate immune cells. Figure 3: Activation of selected splenocyte subpopulations after co-cultivation with 120 s plasma-cells. (**a**) Representative overlay of calcium release assessed via flow cytometry in macrophages and (**b**) T cells, respectively; (**c,d**) quantification of calcium release over time in macrophages and T cells, respectively after plasma treatment (**e**) representative overlay of phosphorylated extracellular signal-regulated Kinase-1/2 (Erk1/2) kinase in T cells 1 h as measured via flow cytometry; (**f**) quantification of Erk1/2 phosphorylation in splenocytes with or without co-culture with 120 s plasma-treated melanoma cells after 1 h or 4 h. Data are mean +S.E. of several spleens; c=control, p=plasma, SPLN=splencocytes. Co-Culture with Plasma-Treated Melanoma Cells Altered the Surface Marker and Cytokine Profile of Murine Immune Cells  
Among splenocytes, CD4\({}^{+}\) T helper and CD8\({}^{+}\) cytotoxic T cells were identified from CD3\({}^{+}\) splenic lymphocytes (Figure 4a). With both lymphocyte subpopulations, the expression of the activation-indicating surface marker CD28 was assessed after co-culture with either untreated melanoma cells or plasma-treated melanoma cells (Figure 4b-c). CD28 expression on the cell surface increased in tendency for CD4\({}^{+}\) and significantly for CD8\({}^{+}\) T cells (Figure 4d). The CD28 cell surface marker provides essential co-stimulation during T cell activation. The expression of other activating T cell markers (CD44, CD152) did not change (data not shown). Only viable cells were included in the analysis, and we also investigated changes of the CD4/CD8 ratio. This parameter is used to describe the immune status, and we found an increase of CD4 over CD8 cells upon co-culture with plasma-treated over non-treated melanoma cells (Figure 4e). This suggests splenic CD4\({}^{+}\) T helper cells survive better in the microenvironment generated by plasma-treated melanoma cells. To assess any changes of myeloid immune cells under these conditions, splenocytes were gated for monocytes and macrophages (Figure 5a).