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

Nova quantificação de ROS usando dihidroetídio em células de câncer de fígado

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.

Autor em destaque: Medição de alto rendimento dos níveis de ROS intracelulares em linhas hepatocelulares

Avaliação de triagem de alto rendimento da geração de espécies reativas de oxigênio (ROS) usando corante de fluorescência Dihidroetídio (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.

Coloração de Diidroetídio (DHE) de Células de Hepatocarcinoma para Quantificação de Espécies Reativas de Oxigênio (EROs)

Para começar, semeie 200 microlitros de volume de 10.000 células de carcinoma hepatocelular em uma placa de 96 poços com DMEM e 10% FBS. Incubar as células durante a noite a 37 graus Celsius em uma incubadora umidificada sob 5% de dióxido de carbono. No dia seguinte, substituir os meios de cultura e tratar as células com concentrações variáveis de peróxido de hidrogênio e menadiona.Dissolva cinco miligramas de DHE em um mililitro de DMSO. Para a solução de trabalho, diluir uma alíquota de 100 micromoles com meio DMEM pré-aquecido para uma concentração de 10 micromoles. Vórtice a solução do corante de trabalho por 5 a 10 segundos para garantir a mistura adequada.Agora remova o meio de teste de cada poço antes de colocar as células do carcinoma. Em seguida, lave suavemente cada poço com PBS. Pipetar 100 microlitros da solução do corante de trabalho em cada poço.Incubar a placa a 37 graus Celsius durante 30 minutos. Remova o meio que contém DHE de cada poço. Use uma pipeta multicanal para lavar cada poço três vezes com PBS.Agora, pipetar 200 microlitros de PBS contendo corante nuclear Hoechst 33342 em cada poço. Após a incubação, transfira a placa para uma plataforma de triagem de alto conteúdo. Inicie o software de triagem de alto conteúdo Cellomics CX7.Calibre a plataforma de imagem de acordo com as especificações do fabricante. Abra os parâmetros do sistema específicos para a marca da placa no banco de dados do instrumento. Em seguida, coloque cuidadosamente a placa com as amostras no estágio aquecido do sistema de imagem.Certifique-se de que a placa está bem posicionada e na orientação correta. Em seguida, pressione suavemente a microplaca para garantir que ela fique encostada no palco. Quando a placa estiver posicionada corretamente, pressione e mantenha pressionada a tecla Control.Em seguida, clique em Controle e OK para abrir a caixa de diálogo Carga/Descarga de placa. Para selecionar os parâmetros de imagem, abra o modo de ativação de destino na interface Cellomics BioApplications. Criar um novo protocolo para aquisição de dados dual-channel compatível com coloração nuclear e sonda de fluorescência DHE.Em seguida, especifique os objetivos necessários para a aquisição da imagem e, em seguida, escolha a ampliação apropriada. Agora especifique o tempo de exposição, a flexão da câmera e o intervalo z-stack. Uma vez definidos os parâmetros de imagem, identifique o objeto de rastreamento primário de interesse usando os diferentes algoritmos do instrumento.Depois de segmentar o objeto identificado, valide o objeto primário com base nos parâmetros específicos de tamanho, forma e intensidade exclusivos para cada tipo de célula. Defina a região de interesse ao redor do objeto e defina um círculo de 20 micrômetros em torno dele. Clique na guia Caracterização da população e, em seguida, defina o limite de varredura para 1000 células por poço com um mínimo de 10 objetos por campo.Agora clique em Launch View para escanear cada placa de poço. Em seguida, inicie o software de análise de visualização e exporte os parâmetros de dados quantitativos em um formato CSV para análise adicional. A exposição ao peróxido resultou em um incremento estatisticamente significativo da fluorescência induzida por ROS em todas as linhagens celulares de forma dose-dependente.Com menadiona, as células JHH-4 apresentaram incremento dose-dependente na intensidade fluorescente. Entretanto, a diferença significativa na fluorescência foi observada apenas em concentrações mais elevadas nas outras duas linhagens 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 ...

Bioengenharia