Quantifying ROS levels using CM-H2DCFDA and HyPer.
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).
Department of Biochemistry, Nencki Institute of Experimental Biology, Warsaw, Poland.
Monika
M
Oparka
Department of Biochemistry, Nencki Institute of Experimental Biology, Warsaw, Poland.
Jarosław
J
Walczak
Department of Biochemistry, Nencki Institute of Experimental Biology, Warsaw, Poland.
Dominika
D
Malinska
Department of Biochemistry (286), RIMLS, RCMM, Radboudumc, Nijmegen, The Netherlands.
Lisanne M P E
LMPE
van Oppen
Department of Biochemistry, Nencki Institute of Experimental Biology, Warsaw, Poland.
Joanna
J
Szczepanowska
Department of Biochemistry (286), RIMLS, RCMM, Radboudumc, Nijmegen, The Netherlands.
Werner J H
WJH
Koopman
Department of Biochemistry, Nencki Institute of Experimental Biology, Warsaw, Poland. Electronic address: m.wieckowski@nencki.gov.pl.
Mariusz R
MR
Wieckowski
D000975:Antioxidants / Q000737:chemistry / Q000302:isolation & purification*; D005452:Fluoresceins / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry; D006861:Hydrogen Peroxide / Q000737:chemistry; D008928:Mitochondria / Q000378:metabolism; D008967:Molecular Biology / Q000379:methods*; D018384:Oxidative Stress; D017382:Reactive Oxygen Species / Q000737:chemistry / Q000302:isolation & purification*
C110400:2',7'-dichlorodihydrofluorescein diacetate; D000975:Antioxidants; D005452:Fluoresceins; D005456:Fluorescent Dyes; D017382:Reactive Oxygen Species; D006861:Hydrogen Peroxide
CM-H(2)DCFDA; HyPer; Hydrogen peroxide; Reactive oxygen species; SypHer