## 3 Results
### Anticancer Efficacy of Frozen PAM  
Next, we evaluated the period of efficacy of PAM. PAM was stored frozen and thawed at the indicated time-points to treat HeLa cancer cells. The results in Figure 5a-b show no significant differences in the effects on HeLa cancer cell viability between fresh and stocked PAM that had been frozen and thawed. The effect was also comparable to that observed for direct plasma treatment. Additionally, we examined whether fresh and stock PAM induced apoptosis in cancer cells after 24 h of treatment with PAM by staining the cells with annexin V conjugated with FITC and PI. After staining, cell death was analyzed by flow cytometry. Apoptosis was detected by labeling phosphatidylserine on the membrane surface of apoptotic cells with annexin V, and late apoptosis/necrosis were detected by labeling cellular DNA with PI. Figure 5c shows that 1- and 6-month stock PAM caused similar levels of cancer cell death and most PAM-treated cancer cells underwent apoptosis.  
Further, we quantitatively assessed the anticancer effects of fresh and stock PAM by diluting PAM and then evaluating its cytotoxic effects on HeLa cells in a live/dead cell assay (Figure 6a) and MTT assay (Figure 6b). Dilutions of PAM by half or 1/4 efficiently induced cell death of HeLa cells but caused slightly lower cell death as compared to undiluted fresh and stock PAM (Figure 6). However, 1/10-diluted PAM did not cause cell death of HeLa cells. Thus, we estimated that the anticancer effect of fresh and stock PAM ranged from 1/10 to 1/4 dilutions. Moreover, we observed a similar dilution  
Figure 4: Comparison of anticancer effects of PAM and direct plasma. (**a**) Human cervical HeLa cancer cells were treated with either PAM or direct plasma, or gas (gas-treatment), stained with calcein-AM and EthD-1, and their viability was examined under a fluorescence microscope. (**b**) Cell viability and activity were assessed by MTT assay and compared to those of untreated or gas-treated control cells. NS, not significant;  
effect between the fresh and stock PAM (Figure 6), indicating that the components required for the anticancer effects in the stock PAM do not decline over time.  
Figure 5: Efficacy of frozen PAM. PAM was stored in a freezer (\(-20\)\({}^{\circ}\)C) for the indicated periods (1 and 6 months), then thawed and added to HeLa cells to evaluate its efficacy. (**a**) Human cervical HeLa cancer cells were treated with plasma (direct plasma or fresh and 1- or 6-month stock PAM) (plasma-treatment) or gas (gas or gas-treated fresh and stock medium) (gas-treatment) for 24 h. Cellular viability was assessed using calcein-AM and EthD-1 to detect live and dead cells, respectively. (**b**) Cell viability was measured by MTT assay and compared to those of gas-treated control cells. (**c**) Cell death via apoptosis/necrosis was detected by staining the cells with FITC-conjugated annexin V and propidium iodide (PI), respectively, and by flow cytometric analysis, and compared to that in gas-treated cells. The dot plots show representative images of apoptotic cell death after PAM-treatment, identified by labeling phosphatidylserine on the membrane surface of early/late apoptotic cells with annexin V, and by labeling DNA of late apoptotic/necrotic cells with PI. The graph shows the populations of apoptosis/necrotic cells.