The Effects of Different Fluorescent Indicators in Observing the Changes of the Mitochondrial Membrane Potential during
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ORIGINAL ARTICLE
The Effects of Different Fluorescent Indicators in Observing the Changes of the Mitochondrial Membrane Potential during Oxidative Stress-Induced Mitochondrial Injury of Cardiac H9c2 Cells Yahan Sun 1 & Kunyan Zhou 2 & Mali He 1 & Ying Gao 1 & Danjie Zhang 1 & Yanwen Bai 1 & Yuezhao Lai 1 & Mengying Liu 1 & Xuechao Han 1 & Sen Xu 1 & Wei Tian 1,3,4 & Jingman Xu 1,3,4 Received: 19 June 2020 / Accepted: 9 September 2020 # Springer Science+Business Media, LLC, part of Springer Nature 2020
Abstract We evaluated the ability of different fluorescent indicators by various analytical instruments, including a laser scanning confocal microscope (LSCM), fluorescence plate reader, and flow cytometer (FCM), to measure the mitochondrial membrane potential (ΔΨm) of cardiac H9c2 cells during oxidative stress-induced mitochondrial injury. The mitochondrial oxygen consumption rate and a transmission electron microscope were used to detect changes in mitochondrial functions and morphology, respectively. Cardiac H9c2 cells were exposed to H2O2 (500, 750, 1000, and 1250 μM) to induce mitochondrial oxidative stress injury, and fluorescent indicators including tetramethyl rhodamine ethyl ester (TMRE), 5,5′,6,6′-tetrachloro-1,1′,3,3′tetraethylbenzimidazolocarbocyanine iodide (JC-1), and rhodamine 123 (R123) were used to detect changes in ΔΨm using an LSCM, fluorescence plate reader, and FCM. The decrease in ΔΨm caused by H2O2 was determined by endpoint and dynamic analyses after staining with JC-1 or TMRE. With the R123 probe, the LSCM could only detect the change in ΔΨm caused by 1000 μM H2O2. Moreover, R123 was less effective than JC-1 and TMRE for measurement of ΔΨm by the LSCM. Our data indicated that an LSCM is the most suitable instrument to detect dynamic changes in ΔΨm, whereas all three instruments can detect ΔΨm at the endpoint. Keywords Mitochondrial membrane potential . Dynamic measurements . Endpoint measurements . Laser scanning confocal microscope . Fluorescence plate reader
Research Highlights R123 is less effective than JC-1 and TMRE for measuring ΔΨm. An LSCM is the most suitable instrument to detect dynamic changes in ΔΨm. * Jingman Xu [email protected] 1
Heart Institute, School of Public Health, North China University of Science and Technology, Tangshan, Hebei, China
2
Department of Cardiovascular, Suning Renmin Hospital, Cangzhou 061000, Hebei Province, China
3
Hebei Province Key Laboratory of Organ Fibrosis, Tangshan, Hebei, China
4
International Scientific and Technological Cooperation Base of Geriatrics Medicine, Tangshan, Hebei, China
Introduction Mitochondria are composed of two layers of membranes, the outer and inner membranes, and are the most important cellular energy-producing organelles. The outer membrane, which is permeable to substances with molecular weights below 10,000 Da, separates the mitochondrial inner space from the cytoplasm, while the inner membrane with a lower permeability divides the mitochondria into the matrix and intermembrane spaces [1] Proton pumps in the
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