## 2 Materials and Methods
### The In Vitro Experiments on Macrophages  
Murine macrophages (RAW264.7) (TIB-71 TM) (ATCC) were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Thermo fisher Scientific, Waltham, MA, USA) and Penicillin-Streptomycin in 5% carbon dioxide (CO\({}^{2}\)) at 37 \({}^{\circ}\)C overnight. After that, the non-stimulated cells were used as a control group. Meanwhile, in the stimulated groups, macrophages at  
Figure 1: Schematic diagram of non-thermal atmospheric pressure plasma. Component of plasma flux generator in schematic diagram (**A**) and the representative pictures of machine (**B**, left side) with an electrode-containing syringe (**B**, middle) and the electrode during plasma flux releasing (**B**, right side) are demonstrated. Cell viability test (MTT assay) in RAW264.7 cells after plasma flux treatment at 30 s at different voltage levels (**C**) and at 10 voltage with different exposure durations (in seconds) (**D**) compared with cells without stimulation (Non) or stimulation with argon gas alone without non-thermal plasma (Argon) are demonstrated. Independent triplicated experiments were performed. 2 \(\times\) 10\({}^{5}\) cell/well were administered by plasma flux or argon gas alone (another control group) with different intensities and durations before cell collection. To determine the proper dose of plasma flux, the cell viability test after plasma flux administration was performed following a published protocol, using tetrazolium dye 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolzolium (MTT) (Thermo fisher Scientific, Waltham, MA, USA) [33]. In brief, the cells in the 96-well plate at the indicated time-points were incubated with a MTT solution in the dark at 37 \({}^{\circ}\)C. After 2 h, the MTT solution was replaced by dimethyl sulfoxide (DMSO; Thermo fisher Scientific, Waltham, MA, USA) and the dissolving purple color was measured by a Varioskan Flash microplate reader (Thermo fisher Scientific, Waltham, MA, USA) at OD 570 nm. With the selected proper dose and duration of the plasma flux treatment, reactive oxygen species (ROS) in macrophages were determined by a dihydroethidium (DHE) fluorescent dye (Sigma-Aldrich, St. Louis, MS, USA) following a previous protocol [34]. In brief, DHE diluted in FBS free media at a concentration of 20 \(\upmu\)M was incubated with the cells at 37 \({}^{\circ}\)C for 20 min before analysis by the Varioskan Flash microplate reader (Thermo fisher Scientific, Waltham, MA, USA) at OD 520 nm. In parallel, the protein abundance of the possible downstream molecules of the ROS stimulation, including AMP-activated protein kinase (\(\alpha\)AMPK) and nuclear factor kappa B (NF-kB), was determined by Western blot analysis following a previous protocol [35]. Briefly, protein was extracted from the samples using a lysis buffer (radioimmunoprecipitation assay buffer; RIPA) in supplement with inhibitors of protease and phosphatase (Thermo fisher Scientific, Waltham, MA, USA). The protein concentration was measured via Bradford assay. Then, the samples were segregated in 10% SDS (sodium dodecyl sulfate) polyacrylamide gel and transferred into the nitrocellulose membrane. Thereafter, several primary antibodies against \(\alpha\)AMPK, phosphorylated \(\alpha\)AMPK, NF-kB p65, phosphorylated NF-kB p65 or the internal control \(\beta\)-actin (Cell signaling, Beverly, MA, USA) were incubated with the membrane prior to horseradish peroxidase (HRP)-conjugated second antibodies and visualized by chemiluminescence (Thermo fisher Scientific, Waltham, MA, USA). The qualification of the band intensity was calculated by Image Studio Lite Ver 5.2 software. To test the effect of plasma flux on inflammatory responses, lipopolysaccharide (LPS), a potent inflammatory stimulator from Gram-negative bacteria, using LPS of _Escherichia coli_ 026: B6 (Sigma-Aldrich) at 100 ng/mL, or media control was added after plasma flux administration.