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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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Profile of RONS on PAM depends on various APPJ operating parameters. Different physical parameters like the gap between APPJ nozzle to cell culture surface (10 or 20 mm), Helium Flow (1, 3, and 5 L/min) and treatment time (5 min) were used to obtain 2 mL of PAM +/− Pyr. (A) Micromolar (μM) concentration of Hydrogen Peroxide (H₂O₂) by AmplexRed/HRP assay. (B) ROS were measured by DCF-DA fluorescence intensity (x10³). (C) Micromolar (μM) concentration of Nitrites (NO₂⁻) by Griess assay. (D) Concentration of NO₂⁻ and H₂O₂ in PAM +/− Pyr at different treatment times (1 to 15 min at 1 L/min and 10 mm gap). PAM + Pyr not lead to significant differences on the concentration of NO₂⁻, with a maximum of 341 ± 27 µM (PAM + Pyr) or 299 ± 10 µM (PAM-Pyr). Data are presented as mean, n = 3, Error bars represent the SD, and asterisks indicate statistically significant differences between the PAM +/− Pyr series (*p < 0.05; **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. The exact concentrations of RONS are presented in Table S2.