TY - JOUR
T1 - Quantifying ROS levels using CM-H2DCFDA and HyPer
AU - Oparka, Monika
AU - Walczak, Jarosław
AU - Malinska, Dominika
AU - van Oppen, Lisanne M.P.E.
AU - Szczepanowska, Joanna
AU - Koopman, Werner J.H.
AU - Wieckowski, Mariusz R.
PY - 2016/10/15
Y1 - 2016/10/15
N2 - 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).
AB - 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).
KW - CM-HDCFDA
KW - Hydrogen peroxide
KW - HyPer
KW - Reactive oxygen species
KW - SypHer
U2 - 10.1016/j.ymeth.2016.06.008
DO - 10.1016/j.ymeth.2016.06.008
M3 - Article
C2 - 27302663
AN - SCOPUS:84994779479
SN - 1046-2023
VL - 109
SP - 3
EP - 11
JO - Methods
JF - Methods
ER -