## 3 Results  
### Plasma Treatment Oxidized THP-1 Monocytes while Being of Modest Toxicity  
Physical plasma is able to generate a broad spectrum of short living reactive species that can be retained as more stable mediators like hydrogen peroxide (H\({}_{2}\)O\({}_{2}\)) in treated liquids. Exposing RPMI cell culture medium to the effluent of the _kINPen_ argon plasma-jet (Figure 1a) generated different amounts of H\({}_{2}\)O\({}_{2}\) in a time-dependent manner that can be described with a polynomial (quadratic) function (Figure 1b). Whereas 20 s of plasma-treatment corresponded to 75 uM H\({}_{2}\)O\({}_{2}\), 120 s generated 300 uM of this oxidative molecule. These regimens were used for treatment of THP-1 monocytes either stimulated with phorbol-12-myristate-13-acetate (PMA) or left unstimulated (no PMA). Redox-sensitive dyes are practical tools to assess intracellular redox state, and two dyes with different emission spectra were used to investigate the cytosolic (DHR123, Figure 1c) and mitochondrial (MTO, Figure 1e) oxidation. For quantification, normalization was done against the control cells that have not received PMA. The treatment with PMA itself did not elicit intracellular oxidation with either dye when compared to the untreated control (Figure 1d,f). By contrast, plasma treatment mediated a significant increase with both probes and independent of PMA stimulation. This suggested plasma-generated ROS reaching the cells and leading to intracellular oxidative reactions, while the change in cytosolic oxidations was more profound compared to mitochondrial reactions. Figure 1: Exposure of THP-1 cells to physical plasma led to oxidation of cytosolic and mitochondrial compartment and altered their metabolic activity. (**a**) A schematic overview of standardized plasma treatment of a 96-well plate via a kINPen argon plasma-jet. (**b**) The concentration of hydrogen peroxide (H\({}_{2}\)O\({}_{2}\)) after plasma treatment of 100 uL RPMI medium with goodness of fit of a polynomial (quadratic) function. (**c**) The representative overlay histogram of DHR123-stained and 120 s plasma-treated or untreated THP-1 cells without PMA and (**d**) its quantification for 0 s, 20 s and 120 s treatment time (\(\pm\)PMA). (**e**) The representative overlay histogram of MTO-stained and 120 s plasma-treated or untreated THP-1 cells without PMA and (**f**) its quantification for 0 s, 20 s and 120 s treatment time (\(\pm\)PMA). (**g**) Metabolic activity 4–6 h post plasma treatment and (**h**) the count of viable (DAPT) THP-1 cells in 1 mL volume, with their calculated mean metabolic activity per cell (blue data points and lines). The data (d and f) are presented as single data points (grey squares) of 4 replicates derived from six independent experiments, and their mean (black bars). The metabolic activity was determined in 8 independent experiments and is presented as (**g**) mean (bar graphs) + SD or (**h**) mean as black bar with replicates as blue boxes accompanied by the mean of activity per cell count (blue data points). The statistical analysis was performed using one-way anova with Dunnett post-testing to respective controls. Asterisks represent statistical comparison to control cells without PMA. The seconds (s) indicate plasma treatment time, ctrl = control. The next question was whether plasma treatment compromised THP-1 cell viability. Four hours following exposure to plasma, the metabolic activity of THP-1 monocytes was measured per well. Quantification revealed only a minor decrease in metabolic activity in non-PMA-pulsed cells (Figure 1g). Similar to oxidation, there was no difference between untreated control and untreated PMA-pulsed cells. Subsequently, absolute cell counts were performed. There was a significant reduction in the total number of viable cells in non-PMA-stimulated THP-1 cells (Figure 1h). In contrast, the viable cell counts of PMA-stimulated cells were largely similar but overall lower compared to non-stimulated cells.