## 6 Viability of A549, A431 and HEK 293 Cells after CAP Jet Treatment
The cell-based experiments were carried out (a) with the grounded substrate under the plate with cells and (b) without a substrate. In Figure 14, the real-time generated outputs from the iCELLigence system before and after direct CAP jets treatment are shown. Cell index was calculated for each E-plate well by RTCA Software 1.2 (Roche Diagnosis, Meylan, France). A dip in Figure 14 refers to the moment when the plate was taken from iCELLigence instrument, irradiated by CAP jet and returned for analysis. Planted cells grow during 24 h before the treatment and 76 h after treatment. iCELLigence data show typical cell index curves that reflect cell proliferation in real-time mode. Figure 14: iCELLigence data showing typical Cell Index curves (CI) that reflect cell proliferation in real-time mode. Data are presented as average of three independent repeats. A549 cells were irradiated without the grounded substrate (**a**) or with it (**b**) for \(U\) = 4.2 kV and \(\
u\) = 5 L/min (helium) or 3.6 kV and \(\
u\) = 3 L/min (argon). The point at which cells were irradiated by CAP is indicated by the arrow. Students t-test was used to compare treatment effects, and the difference between control and groups was statistically significant at \({}^{*}\)\(p\)\(<\) 0.05. For the case without the grounded substrate shown in Figure 14a, the viability of the cells exposed to helium plasma jet is practically the same as the viability of control cells, whereas for the argon plasma jet the number of survivals essentially diminishes. The low efficiency of helium CAP in this case can be explained by a large helium flow rate, v = 5 L/min. The presence of the grounded substrate enhances the effect of plasma treatment (see Figure 14b). For this case, the proliferation rate of the cells lowers to 52 % after the treatment with the helium CAP jet and the argon plasma jet treatment kills almost all cells after 24 h.  
We found that the argon CAP has a greater impact on the cell viability at lower voltages compared to the helium CAP jet. As seen in Figures 13 and 14a, even without grounded substrate, the number of survivals quickly decreases during 48 h. An impact of the argon CAP jet at \(U\) = 3.6 kV looks less aggressive for the case without the grounded substrate, since the cell response begins after several hours after treatment, but not immediately, as with the grounded substrate. It means the \(U\) can be decreased for the former case. To increase an efficiency of the helium CAP we use the optimal parameters for the OH-radical generation found in Section 3 and the grounded substrate beneath the plate with cells proposed in Section 4. According to experimental data shown in Figures 6b and 7, the OH-radical generation rate exhibits a maximum at \(U>\) 4.8 kV and v = 3 L/min. The optimal parameters were applied in the experiment with A549 cells. The plasma jet was generated at \(U\) = 5.5 kV, v = 3 L/min and with the grounded substrate beneath the plates. In Figure 15, the time-dependent viability measured with the iCELigence system is shown before and after the helium CAP treatment. It is seen that after the 2 min and 3 min treatments at the same plasma parameters less than 25% and 2.4% of cells, respectively, survived during 100 h of monitoring. In Figure 15, the data of the indirect CAP treatment is also shown. The media was exposed to the same helium CAP jet during 2 min (with the grounded electrode) and added 15 min after to the wells with cells. The cell index for the case of this indirect treatment practically coincides with the control one. Thus, for indirect treatment the irradiation of media for 2 min, even with the presence of grounded substrate, is insufficient to affect cells. In conclusion of this Section, the indirect CAP jet treatment of A431 and HEK293 cells with the irradiated medium (>5 min) showed various sensitivity of different types of cells. In the direct treatment, the A549 cancer cells were exposed to argon and helium CAP jets at different voltages and gas flow rates.