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. 2019 Mar 26;9:5214. doi: 10.1038/s41598-019-41536-z

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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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Fluorescence intensity of ethidium derived due to the oxidation of DHE by intracellular superoxide (Protocol 1). The fluorescence intensity reflects the levels of intracellular superoxide. The dihydroethidium (DHE) administered in the buffer penetrates cell membrane and is oxidized by superoxide anions to form ethidium (Eth), which emits red fluorescence (excitation filter: 480 nm wavelength; emission filter: 605 nm wavelength). All living cells, which were attached to the bottom of cover glass, were examined for the fluorescence emission after every 10 s. We estimated that the intracellular superoxide level was the highest after pre-conditioning with 13.5 mM glucose + Ang II. Abbreviations: Ang II, angiotensin II; DHE, dihydroethidium; Glu, glucose; mM, mmoL/L; RNH, angiotensin II type 1 receptor antagonist; s, seconds.