Quantifying ROS levels using CM-H 2 DCFDA and HyPer

Methods. 2016 Oct 15;109:3-11. doi: 10.1016/j.ymeth.2016.06.008. Epub 2016 Jun 11.


At low levels, reactive oxygen species (ROS) can act as signaling molecules within cells. When ROS production greatly exceeds the capacity of endogenous antioxidant systems, or antioxidant levels are reduced, ROS levels increase further. The latter is associated with induction of oxidative stress and associated signal transduction and characterized by ROS-induced changes in cellular redox homeostasis and/or damaging effects on biomolecules (e.g. DNA, proteins and lipids). Given the complex mechanisms involved in ROS production and removal, in combination with the lack of reporter molecules that are truly specific for a particular type of ROS, quantification of (sub)cellular ROS levels is a challenging task. In this chapter we describe two strategies to measure ROS: one approach to assess general oxidant levels using the chemical reporter CM-H2DCFDA (5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate), and a second approach allowing more specific analysis of cytosolic hydrogen peroxide (H2O2) levels using protein-based sensors (HyPer and SypHer).

Keywords: CM-H(2)DCFDA; HyPer; Hydrogen peroxide; Reactive oxygen species; SypHer.

Publication types

  • Review
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antioxidants / chemistry
  • Antioxidants / isolation & purification*
  • Fluoresceins / chemistry
  • Fluorescent Dyes / chemistry
  • Hydrogen Peroxide / chemistry
  • Mitochondria / metabolism
  • Molecular Biology / methods*
  • Oxidative Stress*
  • Reactive Oxygen Species / chemistry
  • Reactive Oxygen Species / isolation & purification*


  • 2',7'-dichlorodihydrofluorescein diacetate
  • Antioxidants
  • Fluoresceins
  • Fluorescent Dyes
  • Reactive Oxygen Species
  • Hydrogen Peroxide