Measurement of Reactive Oxygen Species, Reactive Nitrogen Species, and Redox-Dependent Signaling in the Cardiovascular System

Abstract

Reactive oxygen species and reactive nitrogen species are biological molecules that play important roles in cardiovascular physiology and contribute to disease initiation, progression, and severity. Because of their ephemeral nature and rapid reactivity, these species are difficult to measure directly with high accuracy and precision. In this statement, we review current methods for measuring these species and the secondary products they generate and suggest approaches for measuring redox status, oxidative stress, and the production of individual reactive oxygen and nitrogen species. We discuss the strengths and limitations of different methods and the relative specificity and suitability of these methods for measuring the concentrations of reactive oxygen and reactive nitrogen species in cells, tissues, and biological fluids. We provide specific guidelines, through expert opinion, for choosing reliable and reproducible assays for different experimental and clinical situations. These guidelines are intended to help investigators and clinical researchers avoid experimental error and ensure high-quality measurements of these important biological species.

TRPC3-Nox2 complex mediates doxorubicin-induced myocardial atrophy

“…TRPC3 knockdown suppressed this cardiomyocyte atrophy ( Figure 3A) and increased 2',7'-dichlorofluorescein diacetate (DCFH 2 DA) fluorescence intensity ( Figure 3B). Despite the high sensitivity of DCFH 2 DA to ROS, this probe is limited to use for measuring intracellular ROS (31). Therefore, we used a genetically encoded fluorescent sensor for hydrogen peroxide, Hyper, and confirmed that a DOX-induced increase in Hyper fluorescence intensity was suppressed by the treatment with siRNA for TRPC3 or Nox2 in H9c2 cardiac myoblasts (Supplemental Figure 2A).…”

Sex‐Specific Mechanisms of Resistance Vessel Endothelial Dysfunction Induced by Cardiometabolic Risk Factors

“…Interestingly, the reduction in hydrogen peroxide in vessels from hyperlipidemic females was associated with increased ROS generation evaluated by DHE fluorescence. DHE is widely used to detect superoxide generation with high sensitivity, although other oxidants can produce a 2‐electron oxidation product with similar fluorescence characteristics 50. Consequently, the results suggest increased superoxide generation concomitant with reduced hydrogen peroxide in small arteries of female hyperlipidemic mice.…”

A Novel Approach to Estimate ROS Origination by Hyperbaric Oxygen Exposure, Targeted Probes and Specific Inhibitors

“…From the confocal images, it can be seen that nuclei and/or mitochondria were labeled by DHE or Mito-SOX. However, due to the probes' DNA affinity and required higher resolution, precise ROS locations cannot be identified correctly in images [24]. Flow cytometry values had the least variation between repetitions among the three methods.…”

“…The main devices for fluorescence detection are flow cytometer, confocal microscopy and microplate reader, with respective advantages and disadvantages [24]. Flow cytometry is the most popular and sensitive method currently, but necessary steps such as digestion may influence ROS detection.…”