Measurement of Oxidative Stress Markers In Vitro Using Commercially Available Kits

Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are highly reactive molecules, with significant effects in human diseases including cancer and cardiovascular disease. The ability to accurately and precisely detect the formation of free radicals within cells and tissues is crucial to developing proper treatments for the problems caused by ROS/RNS. Fluorescent probes have become widely available reagents of detecting ROS/RNS within cells. Several commercially available kits have shown their specificity toward detecting the formation of ROS and RNS. In this chapter, we discuss the principle behind each kit and the benefits and shortcomings of these kits, namely dihydroethidium (DHE), dichlorohydrofluoresin diacetate (DCF-DA), 4-amino-5-methylamino-2′,7′-difluorofluorescein diacetate (DAF-FM diacetate), and 10-acetyl-3,7-dihyrdroxyphenoxazine (Amplex red). DHE is used to specifically detect superoxide, while DCF-DA readily detects hydroxyl radicals. Amplex red is used to detect hydrogen peroxide, and DAF-FM is used for measuring nitric oxide. However, due to the nature of their reactivity, the probes are not absolutely specific for the noted ROS/RNS species, and will react with others. ROS measurement may need to be made in real time, and they are short-lived within the cell, especially superoxide and nitric oxide. This chapter explains the mechanism behind each chemical kit, the protocols used with the kit, and show typical results after imaging. Additionally, an assessment is made on the use of the kit, identifying the advantages and disadvantages of each probe.