## 3 Results
### Plasma Flux Promoted Wound Healing in Burn Wounds of Mice, Regardless of Infection  
For the in vivo experiments, two mouse models of burn wounds with and without infection by _Staphylococcus aureus_, the most common secondary infection in burn wounds [42], were used. Notably, the susceptibility against plasma flux of _S. aureus_ and methicillin resistant _S. aureus_ (MRSA) were non-different, as an approximately 30% reduction in colony count (by culture at 24 h) in both strains after a 30 s plasma flux exposure was found (data not shown). As such, the macroscopic wound monitoring at 7 days post-injury demonstrated a higher area of the wound with prominent inflammation (wound rank score, see method) in burn wounds with bacterial infection, compared with the non-infected wound (Figure 5A-C). With the plasma flux treatment, the wound area was smaller with fewer prominent inflammatory signs in the burn wound models either with or without bacterial infection (Figure 5A-C). However, our models were not severe enough to demonstrate the systemic effect of burn wounds or infected burn wounds as indicated by the non-infection.  
Figure 3: Responses of plasma-pretreated macrophages (RAW264.7 cells) followed by stimulation with LPS or phosphate buffer saline (PBS) (negative control) at different time-points as evaluated by supernatant cytokines (TNF-\(\alpha\), IL-6) (**A**,**B**), gene expression of M1 macrophage polarization markers (_IL-1B_ and _iNOS_) (**C**,**D**) and M2 macrophage polarization markers (_IL-10, Arg1, Fizz_ and _TGF-\(\beta\)) (**E**–**H**) are demonstrated. Independently triplicated experiments were performed.  
difference in serum cytokines and the gut permeability defect when compared with the control group (Figure 5D-F), which is different from other publications [30, 43]. At 7 days, plasma flux attenuated tissue pro-inflammatory cytokines (TNF-\(\alpha\) and IL-6), but not anti-inflammatory IL-10, in the bacterial infected burn wounds but not in the non-infected burn wounds (Figure 6A-C). These data support an anti-inflammatory effect of plasma during the wound healing process. Despite the bactericidal activity of plasma flux in vitro [44], plasma flux did not reduce bacterial burdens in burn wounds with or without infection (Figure 6D). Moreover, the accumulation of collagen, an indicator of improved wound healing [45], as determined by [PERSON]'s trichrome staining, was higher in plasma-treated non-infected burn wounds compared with the control group (Figure 6E). However, the collagen deposition was not different between the plasma treatment and control group in the infected burn wound model (Figure 6E). These data imply that plasma flux promotes wound healing in infected burn wounds partly through anti-inflammation (skin cytokines) (Figure 6A,B), while improving the wound condition in non-infected burn wounds, at least in part, through accelerated collagen deposition (Figure 6E).  
Figure 4: Responses of fibroblasts (L929 cell line) to plasma treatment (30 s), when compared with cells without stimulation (Non) or stimulated with argon gas alone (Argon) as evaluated by fibroblast migration (fibroblast scratch assay) with the representative microscopic photographs at different time points and scoring comparison (**A**,**B**) with fibroblast proliferation, using carboxyfluorescein diacetate succinimidyl ester (CFDA-SE) fluorescent staining and the representative flow cytometry analysis for CFDA-SE (**C**,**D**) are demonstrated. Independently triplicated experiments were performed.  
Figure 5: Characteristics of wounds in mouse models with burn injury with or without bacterial infection after treatment by plasma flux (Plasma) or Argon gas alone (Untreat) as determined by wound area and wound rank score (see method) with the representative pictures (**A–C**), serum cytokines (IL-6 and TNF-\(\alpha\)) (**D,E**) and gut permeability measurement (FITC-dextran assay) (**F**) (n = 5–7/ time-point or group) are demonstrated.