Concerns in the application of fluorescent probes DCDHF-DA, DHR 123 and DHE to measure reactive oxygen species in vitro

Yazdani, Mazyar

doi:10.1016/j.tiv.2015.08.010

Cited by 74 publications

(48 citation statements)

References 44 publications

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“…Thus, we tested the influence of NOX2 on probe oxidation in E. coli . The generation of reactive species by PLB-985 cells was confirmed using the oxidant-sensitive dye 2',7'-Dichlordihydrofluorescein-diacetate (H 2 DCFDA), which is oxidized intracellularly to 2',7'-Dichlorfluorescein (DCF) by a number of reactive oxygen species ( Chen et al, 2010 ; Yazdani, 2015 ). Neutrophil-like PLB-985 cells, when stimulated with PMA, showed higher DCF fluorescence than non-stimulated cells ( Figure 7A ), confirming previous reports that these cells do indeed produce ROS ( Segal et al, 1980 ).…”

Section: Resultsmentioning

confidence: 97%

Neutrophil-generated HOCl leads to non-specific thiol oxidation in phagocytized bacteria

Degrossoli

Müller

Xie

et al. 2018

eLife

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Phagocytic immune cells kill pathogens in the phagolysosomal compartment with a cocktail of antimicrobial agents. Chief among them are reactive species produced in the so-called oxidative burst. Here, we show that bacteria exposed to a neutrophil-like cell line experience a rapid and massive oxidation of cytosolic thiols. Using roGFP2-based fusion probes, we could show that this massive breakdown of the thiol redox homeostasis was dependent on phagocytosis, presence of NADPH oxidase and ultimately myeloperoxidase. Interestingly, the redox-mediated fluorescence change in bacteria expressing a glutathione-specific Grx1-roGFP2 fusion protein or an unfused roGFP2 showed highly similar reaction kinetics to the ones observed with roGFP2-Orp1, under all conditions tested. We recently observed such an indiscriminate oxidation of roGFP2-based fusion probes by HOCl with fast kinetics in vitro. In line with these observations, abating HOCl production in immune cells with a myeloperoxidase inhibitor significantly attenuated the oxidation of all three probes in bacteria.

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Section: Resultsmentioning

confidence: 97%

Neutrophil-generated HOCl leads to non-specific thiol oxidation in phagocytized bacteria

Degrossoli

Müller

Xie

et al. 2018

eLife

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“…DCF emits green fluorescence which is directly proportional to the amount of oxidized DCFDA to DCF. The maximum excitation and emission spectra are 495 and 529 nm, respectively ( 37 , 38 ). Neutrophils were preloaded with 5 µM of DCFDA dye and either left unstimulated (−ve control) or activated by PMA at different salt and d -mannitol concentrations.…”

Section: Methodsmentioning

confidence: 99%

Hypertonic Saline Suppresses NADPH Oxidase-Dependent Neutrophil Extracellular Trap Formation and Promotes Apoptosis

et al. 2018

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Tonicity of saline (NaCl) is important in regulating cellular functions and homeostasis. Hypertonic saline is administered to treat many inflammatory diseases, including cystic fibrosis. Excess neutrophil extracellular trap (NET) formation, or NETosis, is associated with many pathological conditions including chronic inflammation. Despite the known therapeutic benefits of hypertonic saline, its underlying mechanisms are not clearly understood. Therefore, we aimed to elucidate the effects of hypertonic saline in modulating NETosis. For this purpose, we purified human neutrophils and induced NETosis using agonists such as diacylglycerol mimetic phorbol myristate acetate (PMA), Gram-negative bacterial cell wall component lipopolysaccharide (LPS), calcium ionophores (A23187 and ionomycin from Streptomyces conglobatus), and bacteria (Pseudomonas aeruginosa and Staphylococcus aureus). We then analyzed neutrophils and NETs using Sytox green assay, immunostaining of NET components and apoptosis markers, confocal microscopy, and pH sensing reagents. This study found that hypertonic NaCl suppresses nicotinamide adenine dinucleotide phosphate oxidase (NADPH2 or NOX2)-dependent NETosis induced by agonists PMA, Escherichia coli LPS (0111:B4 and O128:B12), and P. aeruginosa. Hypertonic saline also suppresses LPS- and PMA- induced reactive oxygen species production. It was determined that supplementing H2O2 reverses the suppressive effect of hypertonic saline on NOX2-dependent NETosis. Many of the aforementioned suppressive effects were observed in the presence of equimolar concentrations of choline chloride and osmolytes (d-mannitol and d-sorbitol). This suggests that the mechanism by which hypertonic saline suppresses NOX2-dependent NETosis is via neutrophil dehydration. Hypertonic NaCl does not significantly alter the intracellular pH of neutrophils. We found that hypertonic NaCl induces apoptosis while suppressing NOX2-dependent NETosis. In contrast, hypertonic solutions do not suppress NOX2-independent NETosis. Although hypertonic saline partially suppresses ionomycin-induced NETosis, it enhances A23187-induced NETosis, and it does not alter S. aureus-induced NETosis. Overall, this study determined that hypertonic saline suppresses NOX2-dependent NETosis induced by several agonists; in contrast, it has variable effects on neutrophil death induced by NOX2-independent NETosis agonists. These findings are important in understanding the regulation of NETosis and apoptosis in neutrophils.

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“…[65][66][67][68][69][70][71] More recent approaches use genetically-encoded protein sensors or transgenic organisms to determine H 2 O 2 . [72][73][74][75][76][77][78][79] While these approaches all have advantages and disadvantages, 65,71,[80][81][82][83][84] one of their major problems is the quantification of absolute [H 2 O 2 ] or even cellular [H 2 O 2 ] kinetics. Another serious issue is the fact that measurements with fluorescent proteins may cause phototoxicity leading to oxidative stress in cells.…”

Section: Redox Chemistry Of Hydrogen Peroxidementioning

confidence: 99%

Review—Quantification of Hydrogen Peroxide by Electrochemical Methods and Electron Spin Resonance Spectroscopy

Gulaboski

Mirčeski

Kappl

et al. 2019

J. Electrochem. Soc.

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Reactive oxygen species (ROS) exhibit different spatial and temporal distributions as well as concentrations in-and outside the cell, thereby functioning as signaling or pathogen-destroying molecules. Especially the ROS H 2 O 2 is important for the patho/physiological status of an organism. Electrochemistry (EM) and electron spin resonance (ESR)-based techniques allow quantification of H 2 O 2 in artificial and living systems, coping a concentration range from low nM up to mM. Working electrodes for EM are optimized by diverse modifications and, additionally, redox mediators are used. Ultramicroelectrodes allow scanning of single cells to spatially resolve and quantify extracellular H 2 O 2 in real-time. With ESR spectroscopy, • O 2¯, but not H 2 O 2 , can be directly determined by spin probes in-and outside of cells in suspensions. Monitoring H 2 O 2 requires formation of intermediate radicals, detectable with spin probes. Low μM [H 2 O 2 ] can thus be assessed specifically. Using suitable spin traps, in-vivo ESR and immuno-spin trapping can visualize different radicals at their respective production sites in small animals, organs and tissues. Here, the redox reaction cascades may interfere with cell metabolism. Optimization of all methods established for H 2 O 2 determination would be favorable to finally combine them for mutual validation. Thus, a deeper insight into cellular ROS metabolism can be obtained.

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Concerns in the application of fluorescent probes DCDHF-DA, DHR 123 and DHE to measure reactive oxygen species in vitro

Cited by 74 publications

References 44 publications

Neutrophil-generated HOCl leads to non-specific thiol oxidation in phagocytized bacteria

Neutrophil-generated HOCl leads to non-specific thiol oxidation in phagocytized bacteria

Hypertonic Saline Suppresses NADPH Oxidase-Dependent Neutrophil Extracellular Trap Formation and Promotes Apoptosis

Review—Quantification of Hydrogen Peroxide by Electrochemical Methods and Electron Spin Resonance Spectroscopy

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