Principles of using Cold Atmospheric Plasma Stimulated Media for Cancer Treatment. be the effective reactive species. During the past decade, cold atmospheric plasma (CAP), a near room temperature plasma mainly composed of reactive oxygen species (ROS) and reactive nitrogen species (RNS)1, has been extensively investigated for its promising application in anti-cancer therapy2,3. So far, CAP has shown a significant anti-cancer capacity over a wide range of cancer cell lines, including carcinomas4,5, melanomas6,7, neuroectodermal malignancies8,9, and hematopoietic malignancies10,11. In addition, the CAP also strongly resists tumor growth in mice12,13. Several general conclusions about the anti-cancer mechanism of CAP have been acknowledged. First, the rise of intracellular ROS always occurs in cancer cells upon CAP treatment4,14, which causes a noticeable damage around the antioxidant system15,16 and subsequently DNA double strands break (DSB) to a fatal degree1,17. Second, serious DNA damage and other effect of CAP on L-741626 cancer cells result in the cell cycle arrest18, apoptosis or necrosis with a dose-dependent pattern19,20. Third, among diverse reactive L-741626 species generated in CAP, H2O2 and NO are proposed to be key molecules to kill cancer cells5,21. Fourth, untransformed normal cells always show stronger resistance to CAP than cancer cells do12,22. Such killing preference on cancer cells is always accompanied with the distinct ROS levels and DSB among cancer cells and normal cells23,24. Conventionally, the CAP is usually directly used to irradiate cancer cells or tissue. Over past three years, the CAP irradiated media was also found to kill cancer cells as effectively as the direct CAP treatment did8,25. In contrast to the direct CAP treatment, the CAPs media has advantages. The CAPs media can be stored in the refrigerator and maintain its anti-cancer capacity for at least 7 days26. Thus, the CAPs media might be a good fit for the condition where a CASP8 CAP device is not available. Moreover the CAPs media can be injected into tissues and effectively prevent tumor growth27. These tissues may not be easily penetrated by the CAP jet, which only causes the cell death in the upper 3C5 cell layers of the CAP touched tissues28. To date, the anti-tumor capacity of the CAPs media has been researched less than the direct CAP treatment. Therefore, basic principles to guide its application remain elusive. In this study, four factors have been found to be capable of optimizing the anti-cancer capacity of the CAPs media on glioblastoma cells (U87), breast cancer cells (MDA-MB-231 and MCF-7): the treatment time, the well size, the gap between plasma source and liquid, and the volume of media,. Glioblastoma is the most lethal form of brain cancer29. Due to its strong resistance to conventional therapy, the median survival time of patients is only 15 months29,30. CAP has L-741626 shown promising anti-cancer capacity on glioblastoma cells observed that when an atmospheric-pressure plasma jet touched a glass surface, it flowed radically over the glass surface and formed a large area made up of reactive species around the glass surface40. Thus, the reactive species in the plasma jet should affect an area of liquid that is significantly larger than the diameter of the jet. The half-life of .OH is only a few microseconds41, however, which eliminates the possibility that .OH diffuses over the liquid surface (Fig. S2a). In contrast, H2O2 and NO with much longer half-life may enter the media by the diffusion over the whole surface of liquid. We denote that H2O2/NO area and .OH area to represent the area mainly affected by H2O2/NO and .OH around the liquid surface.