Background

Reactive oxygen species (ROS) can be generated in different cellular compartments such as peroxisomes, the cytosol, at mitochondrial membranes, or at the plasma membrane (Di Meo et al., 2016). High concentrations of ROS can damage all major cellular constituents and are, thus, a lethal threat for living organisms. High amounts of ROS produced in a short period of time (often called oxidative burst) are important for the immune system to fight pathogens. Persistence of excessive ROS production is associated with several diseases such rheumatoid arthritis, diabetes mellitus and neurodegenerative diseases. Low concentrations of superoxide anions (O₂^-) and derivatives thereof, however, have been described to act as essential regulators in cellular signaling cascades (Dröge, 2002). The plasma membrane-bound complex NADPH-oxidase 2 (NOX-2) is one of the main O₂^- producers in phagocytes such as dendritic cells, macrophages, and neutrophils. The active NOX-2 complex is formed by 6 subunits (gp91phox, p22phox, Rac2, p40phox, p47phox and p67phox), which allow a tight regulation of NOX-2 activity. NOX-2 can produce both, low and high levels of O₂^- that are rapidly converted into hydrogen peroxide (H₂O₂; Giardino et al., 2017; Belambri et al., 2018). H₂O₂ is comparatively stable and is responsible for both, intra- and extracellular effector functions. High amounts of NOX-2-derived O₂^- produced by neutrophils in phagosomes are important to kill bacteria via direct oxidative damage. In contrast, low concentrations of H₂O₂ fulfill essential intra- as well as intercellular second-messenger functions for the regulation of, e.g., immune responses (Bienert et al., 2006; Holmdahl et al., 2013). Thus, H₂O₂ were demonstrated to enhance the expression of interleukin-2 in activated T cells (Roth and Dröge, 1987) and to inhibit the secretion of pro-inflammatory cytokines in phagocytes (Jendrysik et al., 2011; Sareila et al., 2011; Singel and Segal, 2016; Bode et al., 2019). Accordingly, loss of functional NOX-2 has been associated with dysregulated immune responses and several autoimmune diseases such as Lupus erythematosus and Crohn’s-like inflammatory bowel disease (Lee et al., 2011; O’Neill et al., 2016; Rosenzweig, 2008; Singel and Segal, 2016).

Several protocols have been established for the detection of free radical production in cells which use either lucigenin, chemiluminescence of luminol, xylenol orange or 2',7'-dichlorodihydrofluorescein diacetate (H₂DCFDA) as tools for the detection of intracellular ROS production (Gyllenhammar, 1987; Dahlgren and Karlsson, 1999; Nourooz-Zadeh, 1999). H₂DCFDA is one of the most frequently used chemicals to investigate the intracellular redox state. Other sensitive and direct approaches for measuring H₂O₂ have also been described such as the Amplex Red Hydrogen Peroxide/Peroxidase Assay Kit (Karakuzu et al., 2019). However, this relatively expensive tool measures extracellular H₂O₂ and might not detect low amounts of intracellular ROS. In contrast, low cost, easy handling, high sensitivity, and the possibility to use it in kinetic approaches are important advantages of H₂DCFDA over other established tools. H₂DCFDA enters cells by diffusion and is converted into the non-permeable derivate H₂DCF, which can then be oxidized by ROS into fluorescent 2’,7’-dichlorofluorescein (DCF). An alternative protocol for measuring ROS using H₂DCFDA has been published by Eruslanov and Kusmartsev (Eruslanov and Kusmartsev, 2010). Of note, H₂DCFDA lacks specificity for O₂^- radicals and instead reacts with several O₂^-/H₂O₂ conversion products such as alkoxyl, carbonate, peroxyl, NO₂^-, as well as OH^- radicals (Ischiropoulos et al., 1999; Bilski et al., 2002; Eruslanov and Kusmartsev, 2010). Moreover, H₂DCFDA is prone for artificial conversion mediated, e.g., by light exposure or by direct or indirect oxidation via cytochrome c released during apoptosis (Kalyanaraman et al., 2012). Hence, the detection of ROS by H₂DCFDA must be carefully controlled to confirm cell-dependent ROS production as well as the enzyme of origin such as NOX-2 (Bilski et al., 2002; Ohashi et al., 2002; Eruslanov and Kusmartsev, 2010). In order to further specify the source and species of the measured ROS, different ROS-specific inhibitors (6-Hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid; Trolox, N-Acetyl cysteine; NAC, and catalase) as well as NOX-2-specific inhibitors (sgp91 ds-tat and GSK2795039) are highly recommended.

The following protocol incorporates the use of specific inhibitors to enable a robust detection of low ROS levels in cell lines (Mono Mac 6 (MM6) cells, Jurkat T cells, etc.) and primary immune cells (Bone marrow-derived dendritic cells, etc.). Next to the use of inhibitors as pre-treatment to differentiate source and species mediated by the ROS-inducer of interest (see Table 1), the general ROS scavengers Trolox or NAC are used to stop the reaction of H₂DCF to DCF at the end of the experimental treatment. The latter ensures that only ROS which are produced during the experimental setting are measured and prevents unwanted conversion not related to the treatment such as responses to stress during washing and the final measurement. By using this protocol, highly reproducible detection of intracellular ROS production in signaling-relevant concentrations can be performed (Bode et al., 2019).