Detection of mitochondria-generated reactive oxygen species in cells using multiple probes and methods: Potentials, pitfalls, and the future

Abstract

Reactive oxygen and nitrogen species (ROS/RNS) such as superoxide (O), hydrogen peroxide, lipid hydroperoxides, peroxynitrite, and hypochlorous and hypobromous acids play a key role in many pathophysiological processes. Recent studies have focused on mitochondrial ROS as redox signaling species responsible for promoting cell division, modulating and regulating kinases and phosphatases, and activating transcription factors. Many ROS also stimulate cell death and senescence. The extent to which these processes occur is attributed to ROS levels (low or high) in cells. However, the exact nature of ROS remains unknown. Investigators have used redox-active probes that, upon oxidation by ROS, yield products exhibiting fluorescence, chemiluminescence, or bioluminescence. Mitochondria-targeted probes can be used to detect ROS generated in mitochondria. However, because most of these redox-active probes (untargeted and mitochondria-targeted) are oxidized by several ROS species, attributing redox probe oxidation to specific ROS species is difficult. It is conceivable that redox-active probes are oxidized in common one-electron oxidation pathways, resulting in a radical intermediate that either reacts with another oxidant (including oxygen to produce O) and forms a stable fluorescent product or reacts with O to form a fluorescent marker product. Here, we propose the use of multiple probes and complementary techniques (HPLC, LC-MS, redox blotting, and EPR) and the measurement of intracellular probe uptake and specific marker products to identify specific ROS generated in cells. The low-temperature EPR technique developed to investigate cellular/mitochondrial oxidants can easily be extended to animal and human tissues.

p53 regulates mitochondrial dynamics by inhibiting Drp1 translocation into mitochondria during cellular senescence

“…Since the triphenylphosphonium ion (TPP + ) group of MitoSOX Red has been reported to interfere with mitochondrial membrane potential, we verified that neither mitochondrial membrane potential nor mitochondrial ROS were influenced by TPP + in our experimental setting (Figure S5C,D). Enhanced ROS generation within the mitochondria was further verified by observing the increased oxidation of Prx3, an endogenous marker for redox status (Figure E) . In addition, we observed that the level of intracellular ROS was increased upon p53 expression (Figure F).…”

“…Enhanced ROS generation within the mitochondria was further verified by observing the increased oxidation of Prx3, an endogenous marker for redox status (Figure 2E). 35 In addition, we observed that the level of intracellular ROS was increased upon p53 expression ( Figure 2F). To avoid possible artifacts resulting from the DCF-DA dye, 37 H 2 O 2 production upon p53 expression was further examined by using another ROS probe, Amplex Red.…”

Mechanisms of lung toxicity induced by biomass burning aerosols

“…For detection with DHE, the cells were incubated with 25 μM DHE for 30 min at 37°C [58]. DHE emission was recorded using flow cytometry with an Ex/Em of 488/575 nm [59,60]. Antimycin A (AA) was used as a positive control.…”

“…DHE is a redox-sensitive probe that has been widely used to detect intracellular superoxide anions. The superoxide anion (O· 2 ) reacts with DHE to form an oxidized product and leads to the enhancement of fluorescence [59,60].…”

Reactive Oxygen Species Produced by Mutated Mitochondrial Respiratory Chains of Entire Cells Monitored Using Modified Microelectrodes

“…Various methodologies have been developed to track or quantify ROS production in cells;, they can be classified mainly into four groups, with relation to the methodological approach applied, i. e. electron spin resonance (ESR) probes, fluorescent probes, genetic sensors or electrochemical methods. Each of these techniques suffers from some intrinsic limitations: low specificity and limited quantitative information have been questioned for the commonly used fluorescent probes ,. Expensive, high‐level research equipment and not trivial sample preparation are the main limitation of ESR methods, while the relevant time required for generation of a genetic tool is a strong drawback for genetic sensors.…”