Measurement of Reactive Oxygen Species (ROS) and Mitochondrial ROS in AMPK Knockout Mice Blood Vessels

Abstract

Reactive oxygen species (ROS) are a group of unstable and highly reactive molecules or free radicals typically generated as by-products of cellular processes involving molecular oxygen. In vascular cells, the excessive ROS generation results in the initiation and progression of cardiovascular diseases (CVD). Therefore, a dynamic, robust, and accurate ROS detection method in the blood vessels is essential for pathophysiological research studies of the cardiovascular system.In this chapter, we describe a fluorescence dye-based detection method for assaying superoxide and mitochondrial superoxide in mouse aorta using dihydroethidium (DHE) and MitoSOX. The protocol includes preparation of frozen aortic tissue sections, monitoring DHE oxidation-derived fluorescence by fluorescence microscopy, and high-performance liquid chromatograph-based analysis of MitoSOX and its oxidation products. For studying the role of AMP-activated protein kinase (AMPK) in the redox regulation, we employed AMPKα2 knockout mice and observed increased superoxide and mitochondrial superoxide levels in the aorta of AMPK knockout mice relative to the wild-type group. This novel ROS detection method will be valuable for investigating the roles of cellular and/or mitochondrial ROS in the pathogenesis of CVDs.

Hypoxia and IF1 Expression Promote ROS Decrease in Cancer Cells

“…The higher ROS content, assessed in IF 1 -silenced cells compared to controls in both normoxia and hypoxia, prompted us to investigate whether these changes were associated with a different rate of mitochondrial superoxide anion production. To this aim, we performed experiments by using the specific and sensitive mitochondria-targeted superoxide probe MitoSOX Red [ 27 ]. Loading cells with the dye revealed a mild but significant increase of the superoxide anion production in IF 1 -silenced cells compared to controls, when cells were exposed to either normoxia (about 61 and 50% top right quadrant cells in IF 1 -silenced and control cells, respectively) or hypoxia (about 43 and 33% top right quadrant cells in IF 1 silenced and control cells, respectively) for 24 h ( Figure 5 B).…”

“…Flow cytometry determination of ROS, the most contributors to total cellular reactive oxidant species, and superoxide anion was performed using a MUSE cytometer (Merk Millipore, Darmstadt, Germany) after loading the cells with either 5 μM CellROX Orange or 5 μM MitoSOX Red [ 25 , 26 , 27 ], respectively. The cells were incubated with each dye for 30 min at 37 °C under both normoxia and hypoxia (0.5% O 2 ) and wells were then washed with HBSS to remove any remaining unincorporated dye.…”

Peripheral Blood Mononuclear Cells and Platelets Mitochondrial Dysfunction, Oxidative Stress, and Circulating mtDNA in Cardiovascular Diseases

“…Thus, a normal balance of ROS is essential for cellular functions; however, once the level of ROS surpasses the standard concentration, cellular damage will result, leading finally to apoptosis and cellular death [54,55]. Therefore, an accurate and potent detection method of ROS is crucial for cardiovascular system studies [56], but ROS measurement with high accuracy is still challenging because of ROS' short half-life [57]. Griendling et al listed all measurement approaches of ROS in detail [58].…”

Mechanism of Vascular Toxicity in Rats Subjected to Treatment with a Tyrosine Kinase Inhibitor

“…The most widely used method to quantify cellular O 2 − in mammalian cells is based on DHE staining. MitoSOX or HPLC are currently being used for mitochondrial O 2 − detection, although one major limitation of the latter is the inability to distinguish the specific cells contributing to ROS production in response to pathological stimuli [42]. In our study, we found an increase in superoxide anion (O 2 − ) content in the aorta of rats treated with Su compared with the control group.…”