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  3. Quantifying ROS levels using CM-H2DCFDA and HyPer

Quantifying ROS levels using CM-H2DCFDA and HyPer

  • Methods. 2016 Oct 15;109:3-11. doi: 10.1016/j.ymeth.2016.06.008.
Monika Oparka 1,  Jarosław Walczak 1,  Dominika Malinska 1,  Lisanne M P E van Oppen 2,  Joanna Szczepanowska 1,  Werner J H Koopman 2,  Mariusz R Wieckowski 3
Affiliations

Affiliations

  • 1 Department of Biochemistry, Nencki Institute of Experimental Biology, Warsaw, Poland.
  • 2 Department of Biochemistry (286), RIMLS, RCMM, Radboudumc, Nijmegen, The Netherlands.
  • 3 Department of Biochemistry, Nencki Institute of Experimental Biology, Warsaw, Poland. Electronic address: [email protected].
PMID: 27302663 DOI: 10.1016/j.ymeth.2016.06.008
Abstract

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.

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