MitoSOX-Based Flow Cytometry for Detecting Mitochondrial ROS

  • Megan E. Kauffman Campbell University Jerry M. Wallace School of Osteopathic Medicine, Buies Creek, NC 27506, USA
  • Melinda K. Kauffman Washington and Lee University, Lexington, VA 24450, USA
  • Kassim Traore Campbell University Jerry M. Wallace School of Osteopathic Medicine, Buies Creek, NC 27506, USA
  • Hong Zhu Campbell University Jerry M. Wallace School of Osteopathic Medicine, Buies Creek, NC 27506, USA
  • Michael A. Trush Department of Environmental Health Sciences, The Johns Hopkins University Bloomberg School of Public Health, Baltimore, MD 21205, USA
  • Zhenquan Jia Department of Biology, University of North Carolina, Greensboro, NC 27412, USA
  • Y. Robert Li Campbell University Jerry M. Wallace School of Osteopathic Medicine, Buies Creek, NC 27506, USA; Department of Biology, University of North Carolina, Greensboro, NC 27412, USA; Virginia Tech‒Wake Forest University School of Biomedical Engineering and Sciences, Blacksburg, VA 24061, USA; Department of Biomedical Sciences and Pathobiology, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061, USA
Keywords: B16-F10 melanoma cells, Chemiluminometry, Flow cytometry, Mitochondrial DNA-deficient cells, Mitochondrial ROS, MitoSOX

Abstract

MitoSOX-based assays are widely used to detect mitochondrial reactive oxygen species (ROS), especially superoxide. To this end, 5 µM MitoSOX is commonly used. In this ROS Protocols article, we described the flow cytometric protocol involving the use of various concentrations of MitoSOX (1, 2.5, 5 µM) for detecting mitochondrial ROS in control and mitochondrial DNA-deficient (MD) melanoma B16-F10 cells. We also compared the MitoSOX-based flow cytometry with lucigenin-derived chemiluminometry for their ability to reliably detect the relative differences in mitochondrial ROS formation in the control and MD cells. Our results suggested that 1 µM, rather than the commonly used 5 µM, appeared to be the optimal concentration of MitoSOX for detecting mitochondrial ROS via flow cytometry.
Published
2016-09-01
How to Cite
Kauffman, M. E., Kauffman, M. K., Traore, K., Zhu, H., Trush, M. A., Jia, Z., & Li, Y. R. (2016). MitoSOX-Based Flow Cytometry for Detecting Mitochondrial ROS. Reactive Oxygen Species, 2(5), 361‒370. Retrieved from https://www.aimsci.com/ros/index.php/ros/article/view/62
Section
ROS Protocols