期刊
NEUROSCIENCE LETTERS
卷 399, 期 3, 页码 206-209出版社
ELSEVIER IRELAND LTD
DOI: 10.1016/j.neulet.2006.01.071
关键词
mitochondrial membrane potential; reactive oxygen species; JC-1; fluorescence; visualization; confocal microscopy
Quantitative and qualitative analysis of both generated reactive oxygen species (ROS) and mitrochondrial membrane potential cannot be detected simultaneously. We here introduce a simple, new double staining method. We have successfully used this for several years utilizing cerium for ROS detection and JC-1 staining to assess the mitochondrial membrane potential. The resultant signals on laser confocal images can be localized in the same cells and can easily quantify them. We used a confocal microscope along with our new, combined staining method to both visualize mitochondrial membrane potential (Delta Psi(m)) and imaged ROS. These were quantified by JC-1 staining and by cerium ions with reflectance in a method modified in our laboratory. To test this double labeling technique we used PC 12 cells subjected to I h hypoxia and 24 h re-oxygenization. We are able to produce a quantitative analysis of red/green signals of JC-1 that reflected the energy state of the cells. Cerium reflectance correlates with the amount of ROS release in the same cells. Significant differences have been calculated after hypoxia and re-oxygenation in both modality of the cell staining. The red/green ratio was 18.2 +/- 9.3 (n = 30) in normoxic cells versus 1.65 +/- 0.9 (n = 30) in the hypoxia/re-oxygenation group (p < 0.05). In the same randomly selected cells the average cerium reflectance signal intensity was 2.5 +/- 1.2 (n = 30) in the control group while 5.8 +/- 3.1 (n = 30) in the hypoxia/re-oxygenation group (p < 0.05). This assay, by characterizing hypoxic injury and re-oxygenization induced ROS production, offers a qualitative and quantitative method to detect the consequences of oxidative stress in experimental conditions and to detect different cell protective strategies. (c) 2006 Elsevier Ireland Ltd. All rights reserved.
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