## 3 Results  
The administration of non-thermal plasma flux promoted the healing process of burn wounds through several wound-healing promotion factors (anti-inflammatory macrophages and improved fibroblast migration). Non-Thermal Plasma Flux Induced Anti-Inflammatory Macrophages and Fibroblast Migration, but Not Fibroblast Proliferation  
An in-house non-thermal plasma flux generator, using argon gas in atmospheric pressure with adjustable plasma properties (electrical frequency and voltage), was developed (Figure 1A,B). Due to the importance of macrophages on the wound healing process, different plasma properties, titrated by voltages and durations, were applied on macrophages (RAW246.7 cell line) before the cell viability test (MTT assay, see method) to determine the proper dose of plasma flux. With the duration of the plasma treatment at 30 s, all of the selected input voltages, in a range between 9.5 and 12.5 volts (V), demonstrated a non-statistical difference on cell viability when compared with the control non-treatment (Non) or argon gas alone without non-thermal plasma (Argon) (Figure 1C). However, the power supply voltage below 10 V showed a tendency of more survived macrophages (Figure 1C). Input with 10 V (corresponding with the output of 10 kV) was applied in the different durations, from 30 s to 90 s, and a duration of less than 30 s demonstrated a decreased number of macrophage cell deaths (Figure 1D). Notably, argon gas alone did not affect the macrophages and only argon with 90 s is shown in Figure 1D. Subsequently, non-thermal plasma with 10 V input (10 kV output) for 30 s was selected for further experiments. Because of the well-known enhanced ROS production by plasma flux [3], ROS in macrophages, using DHE fluorescent dye (see method), was measured. As expected, DHE in the plasma-treated macrophages was higher than the control conditions (Non and Argon) (Figure 2A). \begin{table}
\begin{tabular}{l l l} \hline \multicolumn{1}{c}{**Primers**} & \\ \hline \(\beta\)-_actin_ & Forward & 5\({}^{\prime}\)-CGGTCCAGTGCCCTGAGCTT-3\({}^{\prime}\) \\  & Reward & 5\({}^{\prime}\)-CGTCACACTTCATATGGAATTGA-3\({}^{\prime}\) \\ Inducible nitric oxide synthase (_iNOS_) & Forward & 5\({}^{\prime}\)-CCCTTCCGAAGTTTTCGGCAGC-3\({}^{\prime}\) \\  & Reward & 5\({}^{\prime}\)-GGCTGTCAGAGCCCTGGTGTTG-3\({}^{\prime}\) \\  & Forward & 5\({}^{\prime}\)-CAGAAGAATGGAGAAGCTCAG-3\({}^{\prime}\) \\  & Reward & 5\({}^{\prime}\)-CAGATGGAGAGTCAC-3\({}^{\prime}\) \\  & Forward & 5\({}^{\prime}\)-CGCAGGTCGGAACTTTTIC-3\({}^{\prime}\) \\  & Reward & 5\({}^{\prime}\)-GGACAGGAGGAGGAGG  
In parallel, the possible downstream signals of ROS, including AMPK (a protein sensing cell energy shortage and anti-inflammatory mediator) [39] and NF-kB (a common proinflammatory transcriptional factor) [40] was determined. Accordingly, the abundance of activated NF-kB (ratio of phosphorylated NF-kB-p65/NF-kB-p65), but not AMPK (ratio of phosphorylated \(\alpha\)AMPK/total \(\alpha\)AMPK), decreased after plasma flux administration (Figure 2B-D), implying a modulation of macrophage responses without cell energy alteration. Because of (i) an important transcriptional factor for cytokine production of NF-kB [40] and (ii) a potent pro-inflammatory activating property of lipopolysaccharideacharide (LPS), an organismal molecule [41], the effect of plasma flux on macrophages with or without LPS stimulation was tested. Accordingly, LPS induced pro-inflammatory cytokines (TNF-\(\alpha\) and IL-6) with the peak levels at 24 h post-stimulation (Figure 3A,B). Meanwhile, LPS induced pro-inflammatory macrophage polarization as evaluated by expression of _IL-1_\(\beta\) (peak level at 3-6 h post-LPS) and _iNOS_ (peak level at 24 h Post-LPS) (Figure 3C,D). In parallel, LPS enhanced anti-inflammatory markers, including _IL-10_ (similarly, between 3 and 24 h post-LPS) and _Arg-1_ (peak level at 24 h post-LPS), but not _Fizz_ and _TGF-\(\beta\)_ (Figure 3E-H). Upregulation of anti-inflammatory _IL-10_ was highest in LPS-stimulated macrophages with plasma flux (Figure 3E).