Background

Reactive oxygen and nitrogen species (RONS) is a shared term for oxygen-based radicals and/or chemically reactive oxidants including nitric oxide (•NO), superoxide (O₂•⁻), hydrogen peroxide (H₂O₂), peroxynitrite (ONOO⁻), hypochlorous acid (HOCl), and hydroxyl radical (•OH), among others (Halliwell, 2012). RONS, produced mainly from nicotinamide adenine dinucleotide phosphate oxidase and nitric oxide synthase, play an essential role in signaling cascades regulating cellular processes such as proliferation, differentiation, motility, and programmed cell death (Dröge, 2002). Of note, in view of a cellular vulnerability to oxidants or activators of oxidative metabolism that can induce cell death, an usually tightly regulated steady-state control over the production-detoxification of RONS protects cells from their harmful effects (Harris and Brugge, 2015). This has stimulated increased investigations and lead to tools that are aimed at a better understanding of the biochemistry and physiology of redox reactions of the RONS. However, these tools are not able to assess all facets of redox mechanisms, specifically in living cells and organisms, since detection and quantification of short-lived RONS requires practices that respond to them very rapidly, as opposed to classical antioxidants that finally generate stable products (Dikalov and Harrison, 2014; Brandes et al., 2018). While RONS and redox biology are associated with complex systems, assays that are used for the measurement of RONS involved in biological systems should be approached with caution.

Fluorescent probes produce stable products upon reactions with RONS and can offer high sensitivity detection (Hempel et al., 1999). Redox signaling mechanisms can be explored using 2',7'- dichlorodihydrofluorescein diacetate (DCFH₂-DA); this cell-permeable ester is hydrolyzed inside the cell to 2',7'-dichlorodihydrofluorescein (DCFH₂) by cytosolic esterases, which causes the probe’s retention in the cell to allow detection of intracellular oxidizing reactions (Figure 1). In the presence of RONS, DCFH₂ is oxidized to 2',7'-dichlorofluorescein (DCF) that serves as the fluorescent indicator and can be easily determined, providing a simple and sensitive analytical method for detecting intracellular oxidants (Glebska and Koppenol, 2003). However, oxidation of DCFH₂ in cellular systems reflects not only the rate of production of oxidants and the rate constants for the critical one-electron oxidation step but also other cell-specific and context-dependent aspects. For example, ubiquitous cellular antioxidants such as glutathione (Figure 2) and other protein thiols or antioxidant moieties will attenuate the generation of fluorescence, dependent on the overall redox status (Albrecht et al., 2011). Moreover, despite the popularity of DCFH₂ to assess oxidative stress, the identity of oxidant species responsible remains largely unclear. The correct use and interpretation of the DCFH₂ assay require knowledge about contraindications or pitfalls. Notably, DCFH₂ may be a valuable and sensitive indicator of cellular toxicity, but novel generations of probes must provide information about the exact cellular localization of the oxidizing RONS, reaction rates with different types of RONS, kinetics of the metabolism or decomposition of the oxidized probe, and correlation with downstream signaling (Halliwell and Whiteman, 2004; Winterbourn, 2008; Murphy et al., 2011). Carefully attention to these facts will eliminate erroneous interpretations and help to move exciting approaches of free radical research forward.

Figure 1. Schematic overview of the workflow described in this protocol. CM-DCFH₂-DA diffuses into the cell, intracellular esterases hydrolyze the acetate groups, generating 2',7'- dichlorodihydrofluorescein (DCFH₂) that reacts with intracellular oxidants resulting in the observable highly fluorescent compound 2',7'- dichlorofluorescein (DCF).

Figure 2. Effect of N-acetylcysteine (NAC) and buthionine sulfoximine (BSO) on the formation of DCF. The antioxidant NAC (5 mM) and the pro-oxidant BSO (10 µM), which significantly deplete total glutathione levels by inhibiting γ-glutamyl-cysteine synthetase (a key enzyme in glutathione biosynthesis), were evaluated on human umbilical vein endothelial cells (HUVEC) showing extremes of low and high oxidation of DCFH₂, respectively. HUVEC cells were seeded at the density of 2.0 × 10⁶ cells/cm² for 24 h. BSO and NAC were preincubated for 1 h. The oxidation of DCFH₂ in NAC or BSO treated HUVEC was compared with that of untreated control (CON) cells. The measurements of fluorescence were executed at room temperature. Data were analyzed by one-way analysis of variance with Dunnett’s Multiple Comparison Test. The data represent the means ± SEM. *P < 0.05 versus CON.