## 3 Results and Discussion
### Cell RONS Analysis (Control, 30 s, and 30 s with CAT)  
Cellular RONS were seen to increase under CAP treatment, as shown in Figure 7a. The production of RONS in cells exists in equilibrium with antioxidant defenses. At moderate levels, RONS are believed to be important for regulating physiological and biological functions involved in development, including cell-cycle progression and differentiation, proliferation, and migration. RONS play vital roles in the immune system, maintaining a redox balance, and are associated with the bioactivity of various cell signaling pathways. Excess RONS in cells causes damage to cellular proteins, lipids, nucleic acids, organelles, and membranes that can lead to activation of cell death processes, including cell apoptosis. Apoptosis is a greatly modulated process that is critical for the survival and development of multicellular organisms. These multicellular organisms usually must discard cells that are possibly harmful or superfluous or that possess accumulated mutations. Apoptosis features a representative set of biochemical, pathological, and morphological aspects whereby cells undergo a sequence of self-destruction [34]. We used a cellular RONS analytical method to confirm the CAT and CAP roles on B16F10 cancer cells. Cell RONS levels can be determined in live cells by converting the non-fluorescent 2\({}^{\prime}\),7\({}^{\prime}\)-dichlorofluorescein diacetate (DCFDA) that is oxidized into the fluorescent tracer, 2\({}^{\prime}\), 7\({}^{\prime}\)-dichlorofluorescein (DCF). The generated fluorescent signal is directly proportional to the amount of DCFDA chemically oxidized into DCF. As the fluorescent emission is at the wavelength of ca. 529 nm, it can be determined by fluorescence microscopy, thereby measuring hydroxyl, peroxyl, and other RONS bioactivity in tested cells. Thus, melanoma cells were treated with the transferred CAP in the same way as the aforementioned cell culture method, then cells were stained with DAPI, and DCFDA RONS fluorescence was detected. The fluorescence intensity of RONS was much higher in CAP-treated cells (Figure 7a). The generated cellular RONS signals potentially came from the contributions of applied CAP or cell death after CAP treatment. However, the detailed mechanisms of RONS generation inside cells need to be investigated in the future. At the same time, the group that received CAT, after quantitative plasma treatment and analysis with Image J software (Figure 7b), possessed lower RONS fluorescent signals than the transfer-CAP-treated group without CAT, which confirms the original hypothesis. Furthermore, an interesting research [35], reported that CAP can help in wound healing (non-malignant L929 cells). According to published literature, a moderate ROS level is vital for promoting cell proliferation [36]. A moderate level of ROS plays an essential role in the cell signaling that controls cell survival and cell proliferation. However, a rise in ROS level could damage cell components for example DNA, lipids, proteins, triggering an imbalance among cellular reduction-oxidation (redox) situations and causing homeostasis disruption (in the case of cold-plasma treated cancer cells). By comparing the intensities of the immune-stained CAT level, the staining protein level detected by fluorescence microplate reader in non-malignant L929 cells group was much higher than that in the malignant B16F10 cells cell lines (\(p<0.05\)), which suggests possible higher expression of CAT in L929 cells. The higher CAT expression from L929 cells group might possess an augmented ROS scavenging efficacy, compared to that of the B16F10 cells group, thus causing an anticancer effect when CAP is applied on B16F10 cells (Figure 7c). At present, thus we conclude that CAP can kill B16F10 melanoma cells effectively. Figure 6: **(a)** Fluorescence image of B16F10 cells treated with transferred cold atmospheric plasma (CAP) after 30 s without and with CAT. Green fluorescence represents live cells, while red fluorescence represents dead cells.