The nitrogen atmospheric pressure plasma jet (APPJ) was applied to induce DNA damage of SCC-25 oral cancer cells. Optical emission spectra were taken to characterize the reactive species produced in APPJ. In order to explore the spatial distribution of plasma effects, cells were placed onto photo-etched grid slides and the antibody H2A.X was used to locate double strand breaks of DNA inside nuclei using an immunofluorescence assay. The number of cells with double strand breaks in DNA was observed to be varied due to the distance from the irradiation center and duration of plasma treatment. V

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Shielding-gas-controlled helium atmospheric pressure plasma jets (APPJs) in contact with conductive or nonconductive liquid are investigated. Two-dimensional (2D) optical emission intensity maps of plasma species directly above the liquid, the 2D distribution of reactive oxygen species (ROS) on the liquid surface, and the 2D distribution of DNA double-strand breaks (DSBs) induced in epithelial cancer cells are provided. When conductive liquid is used, the plasma jet develops a glowdischarge-like structure at the liquid surface. The ROS distribution is found to be strongly dependent on both the shielding gas composition and the liquid conductivity. The amount of DNA DSBs is found to be largest when using N 2 shielding gas and almost negligible when using O 2 shielding gas. K E Y W O R D Scell culture, optical emission spectroscopy, plasma jet, plasma treatment, radicals 2 of 15 | KAPALDO ET AL.

The effective clinical application of atmospheric pressure plasma jet (APPJ) treatments requires a well-founded methodology that can describe the interactions between the plasma jet and a treated sample and the temporal and spatial changes that result from the treatment. In this study, we developed a large-scale image analysis method to identify the cell-cycle stage and quantify damage to nuclear DNA in single cells. The method was then tested and used to examine spatio-temporal distributions of nuclear DNA damage in two cell lines from the same anatomic location, namely the oral cavity, after treatment with a nitrogen APPJ. One cell line was malignant, and the other, nonmalignant. The results showed that DNA damage in cancer cells was maximized at the plasma jet treatment region, where the APPJ directly contacted the sample, and declined radially outward. As incubation continued, DNA damage in cancer cells decreased slightly over the first 4 h before rapidly decreasing by approximately 60% at 8 h post-treatment. In nonmalignant cells, no damage was observed within 1 h after treatment, but damage was detected 2 h after treatment. Notably, the damage was 5-fold less than that detected in irradiated cancer cells. Moreover, examining damage with respect to the cell cycle showed that S phase cells were more susceptible to DNA damage than either G1 or G2 phase cells. The proposed methodology for large-scale image analysis is not limited to APPJ post-treatment applications and can be utilized to evaluate biological samples affected by any type of radiation, and, more so, the cell-cycle classification can be used on any cell type with any nuclear DNA staining.

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