Visualization of mitochondrial membrane potential and reactive oxygen species via double staining

Neuroscience Letters
Geza SzilagyiZoltán Nagy

Abstract

Quantitative and qualitative analysis of both generated reactive oxygen species (ROS) and mitochondrial 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 (DeltaPsim) 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 1 h hypoxia and 24h 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+/...Continue Reading

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Citations

Jan 10, 2012·Biological & Pharmaceutical Bulletin·Kwang Sik SuhSuk Chon
Dec 8, 2010·Biochemical and Biophysical Research Communications·Jianqiao LiYan Luo
Aug 6, 2009·The American Journal of Chinese Medicine·Ke YaoYi-Dong Zhang
Feb 28, 2014·International Journal of Molecular Medicine·Woon-Won Jung
Jan 23, 2021·Molecules : a Journal of Synthetic Chemistry and Natural Product Chemistry·Veronika HuntosovaGeorges Wagnieres

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