## 4 Discussion  
Anti-cancer properties of CAP have been recently recognized and are far from being completely understood [55; 56]. In order to enable precise targeting of tumor cells, we  
Figure 5: Comparison of the cytotoxic effect of cold atmospheric plasma in different cell lines. (a) Comparison of the calculated exposure time required to decrease protein content by half (IT50) as assessed by SRB. (b) Comparison of the calculated exposure time required to decrease the metabolic rate by half (IT50) as assessed by MTT. Confidence intervals of 95% are represented by the horizontal bars. The calculated average IC50s of all cell lines are represented as a vertical bar in each figure. *: IT50 not computable. developed an air plasma jet based on a high-voltage sharply-pointed electrode. Plasma is produced according to a point-to-plane pulsed corona discharge model. As the plasma produced by the jet does not significantly increase the local sensible temperature, cooling techniques, which could have serious drawbacks are not needed. Despite the need for thorough quantification, we foresee that such cooling techniques would difficult the plasma application, for example, in endoscopic procedures of non-complacent body cavities. Since plasmas consist of moving electrons and ions, an electric current is created, and they may pass through the skin. The electric characteristics of our device respect the ICNIRP electric current limit for medical applications, defined as 0.5 mA at 1 kHz, and 20 mA at 100 kHz [57]. Then, from this perspective non-intentional contact of plasma created by our device with living tissues, it is not expected to cause any harm. Here, we highlight that a comparison of various experiments carried out with different CAP devices is difficult due to different CAP parameters and designs, as shown in Table 2. Moreover, even assays carried out with the same plasma device rendered divergent results when different cell lines were targeted [21]. In order to allow comparisons between results obtained by different devices, we performed a physical and chemical evaluation of the plasma generated by our equipment. The optical emission spectroscopy (OES) recorded ultraviolet (UV) photons and reactive species emission lines, which are known to induce cellular lesions. UV effects show a strong dependence on dose and wavelength. In fact, the UV absorption spectrum for DNA comprises a rising band in the far UV (<220 nm) with a further peak at 260 nm, which tails into the near-UVB wavelengths at around 300 nm. Between 290 and 315 nm, direct excitation of DNA prevails [58]. However, UV spectral peak wavelengths recorded in our experiment are above the threshold of 300 nm, thus limiting photons' role in plasma cytotoxicity and overemphasizing the role of chemically reactive species. Considering that plasma was generated above a liquid phase where cells were seeded, there are plasma-relevant reactive species that can, by themselves, interact with cancer cells. In fact, most studies of non-thermal plasmas in medicine are based on indirect treatments, with plasmas being produced between two electrodes in a gap discharge or open-air, and ejected into the direction of the sample by convection established by an inert gas flow. This approach has two main implications: (1) there is a gaseous interface between plasma and liquid, which may alter the reactive potential of the generated chemical species, and (2) as reactive species (RS) have to pass through the afterglow area of the plasma jet to reach the surface of the liquid medium, some primary species disappear while other secondary species are generated. In our setup, the device directly delivers plasma to the liquid phase that acts as the grounded electrode. Thus, primary RS are expected to be of greater importance. Table 2 compares the concentrations of nitrites and peroxides found on media exposed to different devices.