Commentary: 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

Yanyi Tian1,4,5, Wei Tian2,4,5, Ting Li3, Jingman Xu1,4,5*

1Heart Institute, School of Public Health, North China University of Science and Technology, Tangshan, Hebei, China

2Analysis and Test Center, North China University of Science and Technology, Tangshan, Hebei, China

3College of Foreign Language, North China University of Science and Technology, Tangshan, Hebei, China

4Hebei Province Key Laboratory of Organ Fibrosis, Tangshan, Hebei, China

5International Scientific and Technological Cooperation Base of Geriatrics Medicine, Tangshan, Hebei, China

This article involves some research results entitled “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”. Myocardial ischemia/reperfusion (MIR) injury, a main pathological manifestation of coronary artery disease, aggravates heart damage after myocardial ischemia, circulatory arrest, or cardiac surgery. Mitochondrial oxidative stress injury is one of the major types of damage caused by MIR injury, while a decrease in the ΔΨm is the earliest indicator of oxidative stress-induced mitochondrial injury1. Therefore, it is very essential to detect changes in ΔΨm accurately and immediately for cardioprotection. Although a previous study has reported that mass spectrometry can be used to detect ΔΨm in animal models, ΔΨm fluorescent indicators, including tetramethylrhodamine ethyl ester (TMRE), 5,5´,6,6´-tetrachloro-1,1´,3,3´-tetraethylbenzimidazolyl carbocyanine iodide (JC-1), and rhodamine 123 (R123), are the most popular agents for ΔΨm detection in single cells and isolated mitochondria2. Meanwhile, many methods have been used to observe the fluorescence intensity of ΔΨm fluorescent indicators in various experimental models, and each has its advantages and disadvantages. In this study, we evaluated the ability of three 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. Among the instruments assessed in this study, the LSCM was the most suitable to detect dynamic changes in ΔΨm, while all three instruments could be used to detect ΔΨm at the endpoint. Furthermore, R123 was less effective than JC-1 and TMRE in measuring ΔΨm by the LSCM.

Although Scaduto RC Jr et al. has reported that the state 3 respiration rates of isolated mitochondria were reduced by TMRE, followed by R1233, the effects of fluorescence indicators on mitochondria is still unclear. Therefore, we tested whether these probes could affect the mitochondrial function and morphology by testing the mitochondrial oxygen consumption rate and a transmission electron microscope, respectively. Our data showed that neither the mitochondrial morphology nor basal respiration was significantly changed by TMRE, JC-1 or R1234. But the maximal respiration decreased markedly after staining cells with these indicators4.

The degree of binding, the sensitivity and the specificity are three indicators of ΔΨm detecting effects evaluation. Each fluorescent probe has its advantages and disadvantages. Although TMRE has less potential-independent binding to cells5,6, it would induce fluorescence quenching when used at high concentrations6. Previous study indicated that JC-1 is a reliable fluorescent probe to assess ΔΨ changes in intact cells comparing to R1237. Recent studies demonstrated JC-1 may not suitable for ΔΨm detecting for the proportion of aggregates in the matrix is distributed red fluorescence and green monomer fluorescence in the cytoplasm are invalid and the relatively hydrophilic probe penetrates the plasma membrane very slowly, which means it may be misled8. The inconvenience in practice also limits the application of the dual-color dye JC-19. In addition, JC-1 has poor water solubility, so it is necessary to repeatedly calibrate the culture concentration in the experiment9. Compared with other dyes, R123 is less photostable10. Therefore, evaluating the effects of different fluorescent indicators on ΔΨm detection may be somewhat confusing. It’s better to compare results obtained with different fluorescent dyes according to a particular experiment before selecting the most accurate probe. S Salvioli et al. demonstrated that TMRE is more effective than R123 to measure mitochondrial depolarization because it can be taken up by live cells rapidly and reversibly11. Whereas JC-1 detects ΔΨm changes in spermatozoa more specifically than other dyes tested, TMRE fluorescence is easily analyzed and these fluorochromes are particularly suitable for multiparametric staining6. JC-1 is a reliable probe for analyzing ΔΨm changes with flow cytometry, while R123 shows a lower sensitivity11. JC-1 appeared to be a more convenient and simple way to detect a functional P-glycoprotein in clinical acute myeloid leukemia (AML) samples than R12311. R123 and JC-1 do not appear to be good probes for the screening of mitochondrial activity in rainbow trout hepatocytes12.

Compared with fluorescence indicators, choosing a suitable instrument is also a key factor in testing ΔΨm. In this study, we evaluated the ability of various analytical instruments, including an LSCM, FCM, in measuring the ΔΨm of cardiac H9c2 cells during oxidative stress-induced mitochondrial injury4. LSCM provides a visual approach to measure ΔΨm by time-dependent scans. Compared with an LSCM, more samples can be observed at one time by a fluorescence plate reader, and all adherent cells can be analyzed simultaneously. FCM can observe multiple cells at one time. Among the instruments assessed in this study, the LSCM was the most suitable to detect dynamic changes in ΔΨm, while all three instruments could be used to detect ΔΨm at the endpoint4. It is necessary to test the damage effect of laser illumination for high-intensity laser illumination may cause photodamage and photobleaching. We tested the effect of laser excitation of an LSCM on cell oxidative stress in our experiments and found the lasers could not increase oxidative damage of the cells in our experimental conditions. Our data implied that TMRE is a suitable fluorescent probe that can be used to detect changes in ΔΨm caused by oxidative stress in live cells by the three instruments, especially the LSCM and fluorescence plate reader. Furthermore, the sensitivity of R123 was less than JC-1 and TMRE to measure ΔΨm by the LSCM.

Besides the ΔΨm losing, oxidative stress may exacerbate the myocardial damage through inducing reactive oxygen species (ROS) generation, calcium overload, endoplasmic reticulum stress and so on13. If we can observe these indicators by staining cells with multiparametric probes simultaneously, it would contribute to many related experiments. Thanks to recently developed instruments and additional probes for cell surface and intracellular markers, detecting ΔΨm along with other biological parameters is used by many researchers14. Previous studies have shown that ΔΨm decreases during the process of cell apoptosis15. For example, Thomas Zuliani et al. proposed reliable and efficient staining, with JC-1 and a fluorescent nuclear acid stain TOTO-3 (1,1'-[1,3-propanediylbis[(dimethyliminio)-3,1-propanediyl]]bis[4-[3-(3-methyl-2(3H)-benzothiazolylidene)-1-propen-1-yl]-,iodide ) to discriminate three functional cellular states: intact, apoptotic, and necrotic/late apoptotic cells by flow cytometry9. The three dyes did not significantly change the mitochondrial morphology and basic respiration, but caused a significant decrease in the maximum respiration value. Studies have shown that (2-(4-(dimethylamino)styryl)-N-ethylpyridinium iodide) DASPEI is also a fluorescent probe for detecting changes in mitochondrial membrane potential, but its limitation is that it can only be applied to the detection of live cell mitochondria15. When choosing an accurate fluorescent probe, it should be selected according to the advantages and disadvantages of the specific experiment combined with the probe. However, the affection among different fluorescent probes including the ROS indicators and calcium indicators  has not been discussed in this paper, which is a limitation.

This article aims to explore the role of different fluorescent indicators in observing changes in mitochondrial membrane potential during oxidative stress mitochondrial damage in cardiac H9c2 cells, and to provide references for the selection of fluorescent probes in related experiments in the future. We should also focus on the relationship between ROS indicator and calcium indicator and fluorescent probe. Therefore, the interaction between fluorescent indicators needs further study.

This work was supported by grant 81700324 from the National Natural Science Foundation of China, grant H2018209378 from the Natural Science Foundation of Hebei Province, grant BJ2018055 from the Hebei Provincial Education Hall top talent project.

  1. Huang DD, Wei XH, Mu HN, et al. Total Salvianolic Acid Injection Prevents Ischemia/Reperfusion-Induced Myocardial Injury Via Antioxidant Mechanism Involving Mitochondrial Respiratory Chain Through the Upregulation of Sirtuin1 and Sirtuin3. Shock. 2019;51(6):745-56. doi:10.1097/SHK.0000000000001185.
  2. Logan A, Pell VR, Shaffer KJ, et al. Assessing the Mitochondrial Membrane Potential in Cells and In Vivo using Targeted Click Chemistry and Mass Spectrometry. Cell Metab. 2016;23(2):379-85. doi:10.1016/j.cmet.2015.11.014.
  3. Scaduto RC, Jr., Grotyohann LW. Measurement of mitochondrial membrane potential using fluorescent rhodamine derivatives. Biophys J. 1999;76(1 Pt 1):469-77. doi:10.1016/S0006-3495(99)77214-0.
  4. Sun Y, Zhou K, He M, et al. 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. J Fluoresc. 2020;30(6):1421-30. doi:10.1007/s10895-020-02623-x.
  5. Ponce WP, Nation JL, Emmel TC, et al. Quantitative analysis of pheromone production in irradiated Caribbean fruit fly males,Anastrepha suspensa (Loew). J Chem Ecol. 1993;19(12):3045-56. doi:10.1007/BF00980601.
  6. Marchetti C, Jouy N, Leroy-Martin B, et al. Comparison of four fluorochromes for the detection of the inner mitochondrial membrane potential in human spermatozoa and their correlation with sperm motility. Hum Reprod. 2004;19(10):2267-76. doi:10.1093/humrep/deh416.
  7. Salvioli S, Ardizzoni A, Franceschi C, et al. JC-1, but not DiOC6(3) or rhodamine 123, is a reliable fluorescent probe to assess delta psi changes in intact cells: implications for studies on mitochondrial functionality during apoptosis. FEBS Lett. 1997;411(1):77-82. doi:10.1016/s0014-5793(97)00669-8.
  8. Nicholls DG. Fluorescence Measurement of Mitochondrial Membrane Potential Changes in Cultured Cells. Methods Mol Biol. 2018;1782:121-35. doi:10.1007/978-1-4939-7831-1_7.
  9. Li X, Zhang R, Guo L, et al. Colocalization Coefficients of a Target-Switchable Fluorescent Probe Can Serve As an Indicator of Mitochondrial Membrane Potential. Anal Chem. 2019;91(4):2672-7. doi:10.1021/acs.analchem.8b03986.
  10. Pfiffi D, Bier BA, Marian CM, et al. Diphenylhexatrienes as photoprotective agents for ultrasensitive fluorescence detection. J Phys Chem A. 2010;114(12):4099-108. doi:10.1021/jp909033x.
  11. Legrand O, Perrot JY, Simonin G, et al. Adult biphenotypic acute leukaemia: an entity with poor prognosis which is related to unfavourable cytogenetics and P-glycoprotein over-expression. Br J Haematol. 1998;100(1):147-55. doi:10.1046/j.1365-2141.1998.00523.x.
  12. Lilius H, Hastbacka T, Isomaa B. A combination of fluorescent probes for evaluation of cytotoxicity and toxic mechanisms in isolated rainbow trout hepatocytes. Toxicol In Vitro. 1996;10(3):341-8. doi:10.1016/0887-2333(96)00015-x.
  13. Xu J, Hao Z, Gou X, et al. Imaging of reactive oxygen species burst from mitochondria using laser scanning confocal microscopy. Microsc Res Tech. 2013;76(6):612-7. doi:10.1002/jemt.22207.
  14. Lugli E, Troiano L, Cossarizza A. Polychromatic analysis of mitochondrial membrane potential using JC-1. Curr Protoc Cytom. 2007;Chapter 7:Unit7 32. doi:10.1002/0471142956.cy0732s41.
  15. Jensen KH, Rekling JC. Development of a no-wash assay for mitochondrial membrane potential using the styryl dye DASPEI. J Biomol Screen. 2010;15(9):1071-81. doi:10.1177/1087057110376834.

Article Info

  • Journal of Cardiology and Cardiovascular Sciences
  • Article Type : Commentary
  • View/Download pdf

Article Notes

  • Published on: January 19, 2021


  • Dynamic measurements

  • Endpoint measurements
  • Fluorescence plate reader
  • Laser scanning confocal microscope
  • Mitochondrial membrane potential


Dr. Jingman Xu
Heart Institute, School of Public Health, North China University of Science and Technology; Hebei Province Key Laboratory of Organ Fibrosis; International Scientific and Technological Cooperation Base of Geriatrics Medicine, Tangshan, Hebei, China;