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Imaging of reactive oxygen species burst from mitochondria using laser scanning confocal microscopy
Author(s) -
Xu Jingman,
Hao Zhimei,
Gou Xiangbo,
Tian Wei,
Jin Yulan,
Cui Shuxia,
Guo Jing,
Sun Yujie,
Wang Ya,
Xu Zhelong
Publication year - 2013
Publication title -
microscopy research and technique
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.536
H-Index - 118
eISSN - 1097-0029
pISSN - 1059-910X
DOI - 10.1002/jemt.22207
Subject(s) - reactive oxygen species , mitochondrion , oxidative stress , membrane potential , intracellular , mitochondrial ros , microbiology and biotechnology , biophysics , chemistry , confocal microscopy , staining , oxidative phosphorylation , confocal , fluorescence , biology , biochemistry , mathematics , physics , quantum mechanics , genetics , geometry
Objective : Although several methods have been used to detect the intracellular reactive oxygen species (ROS) generation, it is still difficult to determine where ROS generate from. This study aimed to demonstrate whether ROS generate from mitochondria during oxidative stress induced mitochondria damage in cardiac H9c2 cells by laser scanning confocal microscopy (LSCM). Methods : Cardiac H9c2 cells were exposed to H 2 O 2 (1200μM) to induce mitochondrial oxidant damage. Mitochondrial membrane potential (ΔΨm) was measured by staining cells with tetramethylrhodamine ethyl ester (TMRE); ROS generation was measured by staining cells with dichlorodihydrofluorescein diacetate (H 2 DCFDA). Results : A rapid/transient ROS burst from mitochondria was induced in cardiac cells treated with H 2 O 2 compared with the control group, suggesting that mitochondria are the main source of ROS induced by oxidative stress in H9c2 cells. Meanwhile, the TMRE fluorescence intensity of mitochondria which had produced a great deal of ROS decreased significantly, indicating that the burst of ROS induces the loss of ΔΨm. In addition, the structure of mitochondria was damaged seriously after ROS burst. However, we also demonstrated that the TMRE fluorescence intensity might be affected by H 2 DCFDA. Conclusions : Mitochondria are the main source of ROS induced by oxidative stress in H9c2 cells and these findings provide a new method to observe whether ROS generate from mitochondria by LSCM. However, these observations also suggested that it is inaccurate to test the fluorescence intensities of cells stained with two or more different fluorescent dyes which should be paid more attention to. Microsc. Res. Tech. 76:612–617, 2013 . © 2013 Wiley Periodicals, Inc.