dihydroethidium &#40;dhe&#41; staining of hepatocarcinoma cells for the quantification of reactive oxygen species &#40;ros&#41;

Nouvelle quantification des ROS à l’aide de dihydroéthidium dans des cellules cancéreuses du foie

Reactive oxygen species (ROS) are a group of naturally occurring, highly reactive, and temporally unstable chemical radicals formed as a part of the normal cellular metabolism in&#160;cells. ROS plays a key and essential role in the modulation of normal physiological and biochemical processes occurring in cells1,2. The main source of ROS production in cells is from the mitochondrial electron transport chain (ETC) pathway as a part of the normal bioenergetic cycle. Significant additional sources of ROS production include enzymatic reactions such as cellular NADPH oxidases in cells. Metabolism of food molecules (e.g., glucose) occurs via the oxidative phosphorylation pathway in the mitochondrial matrix. A baseline level of ROS production is essential to regulate normal physiological cell signaling processes. Many key protein molecules that are part of the glucose metabolic signaling pathways (e.g., Akt and PTEN) are known to respond to intracellular ROS levels. Additionally, ROS are produced by various intracellular enzymes such as xanthine oxidase, nitric oxide synthase, and peroxisomal constituents as a part of the cellular enzymatic pathways1,2. In contrast to the natural sources of ROS, certain environmental factors, such as xenobiotics, infectious agents, UV light, pollution, cigarette smoking, and radiation, also lead to excessive production of ROS, which are a key driver of intra-cellular oxidative stress1,3. Elevated cellular oxidative stress can cause damage to native biomolecules inside a cell, such as lipids, proteins, and DNA, causing various diseases such as cancer, diabetes, cardiovascular disease, chronic inflammation, and neurodegenerative disorders1,3,4. Therefore, accurate measurements of ROS are essential to understand&#160;the cellular mechanisms involved in oxidative stress-induced disease pathophysiology.
Due to the short timescales of ROS production and elimination inside cells, quantitative measurements of various ROS radicals remains a challenge. Methods such as electron paramagnetic resonance (EPR)5, high-pressure liquid chromatography (HPLC), and fluorescence probe-based imaging are used to measure the various cellular ROS6. While methods such as EPR and HPLC yield quantitatively accurate estimates, these methods involve the destruction of the cellular spatial morphology and are usually in the form of global and bulk measurements of a sample. In contrast, imaging-based methods such as fluorescence probe-based methods retain the cellular morphology and spatial context of the ROS generation. However, the specificity of various fluorescence probes for different types of ROS radicals has not been well-established7,8. Several fluorescent probes such as dihydroethidium (DHE), dichlorodihydrofluorescein diacetate (DCFH-DA), dihydrorhodamine (DHR), dimethyl anthracene (DMA), 2,7 dichlorodihydroflurescein (DCFH), 1,3-Diphenylisobenzofuran (DPBF), and MitoSox are available for ROS detection commercially. In the past decades, DHE, MitoSox, and DCFH-DA are the commonly used fluorescent dyes to measure ROS in cells and tissues8,9. DCFH-DA is a widely used dye for detecting intracellular H2O2 and oxidative stress. Despite the popularity of DCFH-DA, multiple previous studies have shown that it cannot be reliably used to measure intracellular H2O2 and other ROS levels8,9,10,11,12,13,14.
In contrast, the fluorescent probe dihydroethidium (DHE) shows a specific response to the intra-cellular superoxide radical (O2-). While the superoxide radical is one of many of the ROS species observed in cells, it is an important radical involved in the reduction of transition metals, conversion to peroxynitrate, and formation of hydroperoxides, among other intracellular effects. DHE is quickly taken up by the cells and has a fluorescence emission in the red wavelength range15. Upon reaction with superoxide radical specifically, DHE forms a red fluorescent product, 2-hydroxy ethidium (2-OH-E+). Thus, DHE may be considered as a specific probe for superoxide detection. However, DHE can also undergo nonspecific oxidation with ONOO- or OH., H2O2, and cytochrome c to form a second fluorescence product, ethidium E+, which can interfere with the measured 2-OH-E+ levels. However, these 2-OH E+ and E+ products, in combination, represent a major part of the total cellular ROS species observed inside a cell when stained with DHE. E+ intercalates into DNA, greatly enhancing its fluorescence8,9,10,11,13,14,15,16. Since the fluorescence spectra of ethidium and 2-hydroxy ethidium only differ slightly, the majority of ROS levels seen in a cell secondary to superoxide production can be detected and measured using DHE fluorescence products. These ROS species are identified using 480 nm wavelength excitation and 610 nm wavelength emission15,16,17.
In addition to choosing a specific fluorescent ROS detection probe, it is important to choose a sensitive method of detection to measure intracellular ROS. Accurate assessment of intracellular ROS levels is thus key to identifying disturbed redox balance states occurring in diseased cells or cells that have been exposed to various environmental stressors such as radiation, toxicological compounds, and genotoxic agents18. Since ROS is a commonly occurring phenomenon in cells that is responsible for regulating a variety of cell signaling activities, robust methods of detection of ROS are essential. To enable such high-throughput evaluation of ROS production within cells, this protocol uses a high-content screening (HCS) platform to measure the ROS species. The current protocol allows the high-throughput analysis of intracellular ROS production, which is of critical importance in many toxicology studies19. This protocol aims to provide an easy and versatile solution to detect and measure intracellular ROS in adherent hepatocellular carcinoma cells. The chemical reagents of H2O2 and menadione are used as potent ROS stimulators to measure the relative levels of ROS production in a controlled and high throughput setting. This protocol may be fine-tuned to measure ROS production in adherent and nonadherent cells under appropriate conditions, as necessary.

Pleins feux sur l’auteur : Mesure à haut débit des niveaux de ROS intracellulaires dans les lignées hépatocellulaires

Évaluation préalable à haut débit de la génération d’espèces réactives de l’oxygène (ROS) à l’aide d’un colorant de fluorescence à base de dihydroéthidium (DHE)

Our lab studies the impact of environmental pollutants, like heavy metals, on liver function and diseases related to reactive oxygen species production. This protocol aims to quantitatively measure ROS levels in cells using a dihydroethidium fluorescence probe. This protocol uses a high-content screening platform to quantitatively measure intracellular ROS species levels in hepatocellular lines.Measuring intercellular ROS level accurately is of critical importance in high-throughput toxicology study, which is a key area of interest in our lab. DHE fluorescence is a highly specific method for measuring total ROS levels, particularly targeting the superoxide radical, are giving it a significant advantage over other commonly used ROS fluorescence probe. Additionally, this protocol offer the unique ability to measure total cellular ROS level in a high-throughput platform.Our protocol is a robust and reproducible choice to characterize intracellular total ROS production. High-throughput approaches to measure ROS levels are helpful in a variety of studies, such as toxicology, drug screening, and cancer biology, which will be our focus in the future.

Coloration au dihydroéthidium (DHE) des cellules d’hépatocarcinome pour la quantification des espèces réactives de l’oxygène (ROS)

Pour commencer, ensemencez un volume de 200 microlitres de 10 000 cellules de carcinome hépatocellulaire dans une plaque de 96 puits avec DMEM et 10 % FBS. Incuber les cellules pendant la nuit à 37 degrés Celsius dans un incubateur humidifié sous 5% de dioxyde de carbone. Le lendemain, remplacez le milieu de culture et traitez les cellules avec des concentrations variables de peroxyde d’hydrogène et de ménadione.Dissoudre cinq milligrammes de DHE dans un millilitre de DMSO. Pour la solution de travail, diluer une aliquote de 100 micromoles avec un milieu DMEM préchauffé à une concentration de 10 micromoles. Agitez la solution de teinture pendant 5 à 10 secondes pour assurer un bon mélange.Retirez maintenant le milieu de test de chaque puits avant de placer les cellules du carcinome. Ensuite, lavez délicatement chaque puits avec du PBS. Pipeter 100 microlitres de la solution de colorant de travail dans chaque puits.Incuber l’assiette à 37 degrés Celsius pendant 30 minutes. Retirez le milieu contenant du DHE de chaque puits. Utilisez une pipette multicanaux pour laver chaque puits trois fois avec du PBS.Maintenant, pipetez 200 microlitres de PBS contenant le colorant de coloration nucléaire Hoechst 33342 dans chaque puits. Après l’incubation, transférez la plaque sur une plate-forme de criblage à haut contenu. Lancez le logiciel de criblage à haut contenu Cellomics CX7.Calibrez la plate-forme d’imagerie selon les spécifications du fabricant. Ouvrez les paramètres système spécifiques à la marque de la plaque dans la base de données de l’instrument. Ensuite, placez soigneusement la plaque contenant les échantillons sur la platine chauffée du système d’imagerie.Assurez-vous que la plaque est bien positionnée et dans la bonne orientation. Appuyez ensuite doucement sur la microplaque pour vous assurer qu’elle repose à plat contre la platine. Une fois la plaque correctement positionnée, appuyez sur la touche Control et maintenez-la enfoncée.Cliquez ensuite sur Contrôle et OK pour ouvrir la boîte de dialogue Chargement/Déchargement de la plaque. Pour sélectionner les paramètres d’imagerie, ouvrez le mode Activation de la cible dans l’interface de Cellomics BioApplications. Créer un nouveau protocole d’acquisition de données à double canal compatible avec la coloration nucléaire et la sonde de fluorescence DHE.Ensuite, spécifiez les objectifs requis pour l’acquisition d’images, puis choisissez le grossissement approprié. Spécifiez maintenant le temps d’exposition, la flexion de la caméra et l’intervalle z-stack. Une fois les paramètres d’imagerie définis, identifiez l’objet de suivi principal d’intérêt à l’aide des différents algorithmes de l’instrument.Après avoir segmenté l’objet identifié, validez l’objet principal en fonction des paramètres spécifiques de taille, de forme et d’intensité propres à chaque type de cellule. Définissez la région d’intérêt autour de l’objet et définissez un cercle de 20 micromètres autour de lui. Cliquez sur l’onglet Caractérisation de la population, puis définissez la limite de numérisation à 1000 cellules par puits avec un minimum de 10 objets par champ.Cliquez maintenant sur Lancer la vue pour numériser chaque plaque de puits. Lancez ensuite le logiciel d’analyse de vue et exportez les paramètres de données quantitatives au format CSV pour une analyse supplémentaire. L’exposition au peroxyde a entraîné une augmentation statistiquement significative de la fluorescence induite par les ROS dans toutes les lignées cellulaires de manière dose-dépendante.Avec la ménadione, les cellules JHH-4 ont montré une augmentation dose-dépendante de l’intensité fluorescente. Cependant, la différence significative de fluorescence n’a été observée qu’à des concentrations plus élevées dans les deux autres lignées cellulaires.

Research

JoVE Journal

Bioengineering

Dihydroethidium &#40;DHE&#41; Staining of Hepatocarcinoma Cells for the Quantification of Reactive Oxygen Species &#40;ROS&#41;

High Throughput Screening Assessment of Reactive Oxygen Species (ROS) Generation using Dihydroethidium (DHE) Fluorescence Dye

Reactive oxygen species (ROS) play a key role in the regulation of cellular metabolism in physiological and pathological processes. Physiological ROS ...

Recherche

Bio-ingénierie