MitoSOX-Based Flow Cytometry for Detecting Mitochondrial ROS

  • React Oxyg Species (Apex). 2016;2(5):361-370. doi: 10.20455/ros.2016.865.
Megan E Kauffman  1 ,  Melinda K Kauffman  2 ,  Kassim Traore  1 ,  Hong Zhu  1 ,  Michael A Trush  3 ,  Zhenquan Jia  4 ,  Y Robert Li  1  4  5  6
Affiliations
  • 1. Campbell University Jerry M. Wallace School of Osteopathic Medicine, Buies Creek, NC 27506, USA.
  • 2. Washington and Lee University, Lexington, VA 24450, USA.
  • 3. Department of Environmental Health Sciences, The Johns Hopkins University Bloomberg School of Public Health, Baltimore, MD 21205, USA.
  • 4. Department of Biology, University of North Carolina, Greensboro, NC 27412, USA.
  • 5. Virginia Tech-Wake Forest University School of Biomedical Engineering and Sciences, Blacksburg, VA 24061, USA.
  • 6. Department of Biomedical Sciences and Pathobiology, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061, USA.
PMID: 29721549 DOI: 10.20455/ros.2016.865
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.

Keywords
B16-F10 melanoma cells; Chemiluminometry; Flow cytometry; MitoSOX; Mitochondrial DNA-deficient cells; Mitochondrial ROS.