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. 2019 Jul 23;9:10681. doi: 10.1038/s41598-019-47128-1

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© The Author(s) 2019

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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H₂O₂ scavengers protect osteosarcoma cells from PAM-induced apoptosis. (A) Effects of PAM +/− Cat activated during 5 minutes (3 L/min helium flow at 10 mm gap) were detected by WST-1 assay on SaOS-2, measured 2, 4 and 24 h after exposure. Catalase was added as control to untreated cell culture medium. Cell viability of Control + Cat, PAM + Cat and PAM-Cat is expressed relative to Control-Cat (untreated) and is the mean and standard deviation of three independent experiments (**p < 0.005; ***p < 0.001; two-sided Student’s t-test). (B) Concentration of NO₂⁻ and H₂O₂ in PAM+/-Catalase at different treatment times (1 to 5 min at 3 L/min and 10 mm gap). Catalase addition to PAM, ⁻not lead to significant differences on the concentration of NO₂⁻, but reduces H₂O₂ concentration in all conditions studied. Data are presented as mean, n = 3, Error bars represent the SD, and asterisks indicate statistically significant differences between the PAM +/− Catalase series (**p < 0.01; ***p < 0.001; two-sided Student’s t-test). Concentration of RONS was measured immediately after APPJ treatments using untreated DMEM as blank. (C) SaOS-2 cells were treated with PAM + Pyr (top) and PAM-Pyr (bottom) activated by plasma at different Helium flows (1, 3 or 5 L/min) during 5 minutes. Apoptosis/Necrosis activation were examined for Annexin V/PI binding using FACS analysis. PAM + Pyr at 1 L/min produces 2.6% apoptotic cells, progressively increasing with 3 L/min (5.5%) and to 5 L/min (29.8%). PAM-Pyr boosts these values, leading to 20% at 1 L/min, 89% at 3 L/min and 93% at 5 L/min of apoptosis positive cells Representative data at 24 hours are shown. Analysis were done in duplicates.