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

Novel ROS Quantification Using Dihydroethidium in Liver Cancer Cells

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.

Author Spotlight: High-Throughput Measurement of Intracellular ROS Levels in Hepatocellular Lines

Evaluación de cribado de alto rendimiento de la generación de especies reactivas de oxígeno (ROS) mediante colorante de fluorescencia de dihidroetidio (DHE)

Tinción de dihidroetidio (DHE) de células de hepatocarcinoma para la cuantificación de especies reactivas de oxígeno (ROS)

Para empezar, siembre un volumen de 200 microlitros de 10.000 células de carcinoma hepatocelular en una placa de 96 pocillos con DMEM y 10% de FBS. Incubar las células durante la noche a 37 grados centígrados en una incubadora humidificada con un 5% de dióxido de carbono. Al día siguiente, reemplace el medio de cultivo y trate las células con concentraciones variables de peróxido de hidrógeno y menadiona.Disuelve cinco miligramos de DHE en un mililitro de DMSO. Para la solución de trabajo, diluir una alícuota de 100 micromoles con medios DMEM precalentados hasta una concentración de 10 micromoles. Agite la solución de tinte de trabajo durante 5 a 10 segundos para garantizar una mezcla adecuada.Ahora retire el medio de prueba de cada pocillo antes de colocar las células de carcinoma. A continuación, lave suavemente cada pocillo con PBS. Pipetear 100 microlitros de la solución de colorante de trabajo en cada pocillo.Incubar la placa a 37 grados centígrados durante 30 minutos. Retire el medio que contiene DHE de cada pocillo. Utilice una pipeta multicanal para lavar cada pocillo tres veces con PBS.Ahora, pipetee 200 microlitros de PBS que contienen el colorante de tinción nuclear Hoechst 33342 en cada pocillo. Después de la incubación, transfiera la placa a una plataforma de cribado de alto contenido. Inicie el software de cribado de alto contenido Cellomics CX7.Calibre la plataforma de imágenes según las especificaciones del fabricante. Abra los parámetros del sistema específicos de la marca de la placa en la base de datos de instrumentos. A continuación, coloque con cuidado la placa con las muestras en la platina calentada del sistema de imágenes.Asegúrese de que la placa esté bien colocada y en la orientación correcta. A continuación, presione suavemente la microplaca para asegurarse de que queda plana contra la platina. Una vez que la placa esté colocada correctamente, mantenga presionada la tecla Control.A continuación, haga clic en Control y Aceptar para abrir el cuadro de diálogo Carga/Descarga de placas. Para seleccionar los parámetros de imagen, abra el modo de activación de objetivos en la interfaz de Cellomics BioApplications. Crear un nuevo protocolo para la adquisición de datos de doble canal compatible con la tinción nuclear y la sonda de fluorescencia DHE.A continuación, especifique los objetivos necesarios para la adquisición de imágenes y, a continuación, elija el aumento adecuado. Ahora especifique el tiempo de exposición, la flexión de la cámara y el intervalo de pila z. Una vez establecidos los parámetros de imagen, identifique el objeto de seguimiento primario de interés utilizando los diferentes algoritmos del instrumento.Después de segmentar el objeto identificado, valide el objeto principal en función de los parámetros específicos de tamaño, forma e intensidad únicos para cada tipo de célula. Defina la región de interés alrededor del objeto y establezca un círculo de 20 micrómetros a su alrededor. Haga clic en la pestaña Caracterización de la población y, a continuación, establezca el límite de escaneo en 1000 celdas por pocillo con un mínimo de 10 objetos por campo.Ahora haga clic en Vista de inicio para escanear cada placa de pocillo. A continuación, inicie el software de análisis de vistas y exporte los parámetros de datos cuantitativos en formato CSV para un análisis adicional. La exposición al peróxido dio lugar a un incremento estadísticamente significativo de la fluorescencia inducida por ROS en todas las líneas celulares de forma dependiente de la dosis.Con la menadiona, las células JHH-4 mostraron un incremento dependiente de la dosis en la intensidad fluorescente. Sin embargo, la diferencia significativa en la fluorescencia se observó solo a concentraciones más altas en las otras dos líneas celulares.

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 ...

Investigación

Bioingeniería