## 3 Results
### Plasma Treatment Altered Surface Marker Expression in THP-1 Cells and Primary Monocytes  
To characterize the cellular phenotype induced with plasma treatment in more detail, the authors screened the membrane expression levels of 14 different surface cell markers and the release of 13 different molecules into the cell culture supernatant. The data summary of changes in the surface  
Figure 3: High content image quantification of morphological changes in THP-1 monocytes up to four days after plasma and/or PMA treatment. 5000 cells were seeded, plasma-treated, and/or PMA-pulsed, and imaged on d0 (4 h), d2 (24 h), and d4 (96 h), respectively. **(a)** The representative images on day 4 of untreated control cells (I), 120 s plasma-treated cells (II) and cells that were exposed to PMA alone (III). The yellow arrows point to differentiated cells with cellular extensions; **(b)** Quantification of cell roundness and, **(c)** quantification of total cell cluster area. Approximately 10,000 single cells per treatment condition were analyzed via computer algorithms. The data are presented as the mean + SEM of three independent experiments with four technical replicates each. The scale bars = 100 \(\upmu\)m. Seconds (s) indicate plasma treatment time, ctrl = control. Figure 2: The validation of the oxidizing effect of plasma and H\({}_{2}\)O\({}_{2}\) in primary monocytes. **(a,b)** The representative overlay histograms of monocytes stained with DHR-123 or MTO, which were treated with 20 s plasma or 70 \(\upmu\)M hydrogen peroxide (H\({}_{2}\)O\({}_{2}\)) in the presence or absence of the antioxidant N-acetyl cysteine (NAC). The geometric mean of the histograms of untreated cells is indicated as black line. **(c)** Normalization of mean fluorescence intensity of DHR-123 and **(d)**_Mitrotacker Orange_ (MTO) stained cells. The data **(c,d)** are representatives out of two independent experiments and show the mean and SD. The seconds (s) indicate plasma treatment time, ctrl = control. marker expression in THP-1 monocytes is given in the form of a heat-map (Figure 4a). The fold change compared to the untreated control samples is shown. As expected, striking changes were found for PMA versus naive cells, for example, a prominent increase in CD11b, CD11c, CD45 RA, CD55, CD63, and CD69 as well as a decrease in CD49d expression. In PMA-pulsed cells, especially CD11b, CD55, CD63, and CD69 were significantly modulated with plasma treatment. In non-stimulated (naive) cells, 120 s of plasma treatment gave a significant increase in CD11c as well as CD55, CD271, and HLA-ABC (Figure 4b-g). Membrane CD55 (Figure 4b) is upregulated through plasma exposure to a level close to the PMA-stimulated group (Figure 4e), while CD271 expression (Figure 4c) even exceeded that with PMA-stimulated samples (Figure 4f). The increase of HLA-ABC (Figure 4d) was generally modest but significant, also in PMA-treated cells (Figure 4g). In primary monocytes, a subset of markers partially different from THP-1 monocytes is described in the literature to monitor activity. Plasma treatment of naive cells did not significantly increase (as in THP-1 cells) but decreased expression levels of CD11c at day 4 (Figure 4h). While levels of CD11b and CD206 were unchanged, a treatment time-dependent and significant increase of CD80 and CD163 was observed. The addition of NAC was demotivated by its ability to change metabolic activity and surface marker profiles of primary monocytes (Supplementary Figure S1c-h). Figure 4: The cell surface marker expression in THP-1 cells and primary monocytes four days after plasma treatment. (**a**) The change of surface marker expressions of untreated and 120 s plasma-treated THP-1 cells with or without PMA in comparison to untreated control cells, displayed as a heat-map (white = decrease, light grey = no change, grey = upregulation, black = upregulation with fold change \(>\)3.