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

Nuova quantificazione dei ROS utilizzando il diidroetidio nelle cellule tumorali del fegato

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.

Autore in primo piano: Misurazione ad alto rendimento dei livelli intracellulari di ROS nelle linee epatocellulari

Valutazione dello screening ad alto rendimento della generazione di specie reattive dell'ossigeno (ROS) utilizzando il colorante a fluorescenza diidroetidio (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.

Colorazione con diidroetidio (DHE) di cellule di epatocarcinoma per la quantificazione delle specie reattive dell'ossigeno (ROS)

Per iniziare, seminare un volume di 200 microlitri di 10.000 cellule di carcinoma epatocellulare in una piastra da 96 pozzetti con DMEM e 10% FBS. Incubare le cellule per una notte a 37 gradi Celsius in un'incubatrice umidificata al 5% di anidride carbonica. Il giorno successivo, sostituire il terreno di coltura e trattare le cellule con concentrazioni variabili di perossido di idrogeno e menadione.Sciogliere cinque milligrammi di DHE in un millilitro di DMSO. Per la soluzione di lavoro, diluire un'aliquota di 100 micromoli con terreno DMEM preriscaldato fino a una concentrazione di 10 micromoli. Agitare la soluzione colorante di lavoro per 5-10 secondi per garantire una corretta miscelazione.Ora rimuovere il terreno di prova da ciascun pozzetto prima di posizionare le cellule di carcinoma. Quindi lavare delicatamente ogni pozzetto con PBS. Pipettare 100 microlitri della soluzione colorante di lavoro in ciascun pozzetto.Incubare la piastra a 37 gradi Celsius per 30 minuti. Rimuovere il terreno contenente DHE da ciascun pozzetto. Utilizzare una pipetta multicanale per lavare ogni pozzetto tre volte con PBS.A questo punto, pipettare 200 microlitri di PBS contenente il colorante colorante nucleare Hoechst 33342 in ciascun pozzetto. Dopo l'incubazione, trasferire la piastra su una piattaforma di screening ad alto contenuto. Avvia il software di screening ad alto contenuto Cellomics CX7.Calibrare la piattaforma di imaging secondo le specifiche del produttore. Aprire i parametri di sistema specifici per la marca della piastra nel database dello strumento. Successivamente, posizionare con cautela la piastra con i campioni sul tavolino riscaldato del sistema di imaging.Assicurarsi che la piastra sia posizionata saldamente e con l'orientamento corretto. Quindi premere delicatamente sulla micropiastra per assicurarsi che poggi piatta contro il tavolino. Una volta posizionata correttamente la piastra, tenere premuto il tasto Ctrl.Quindi fare clic su Controllo e OK per aprire la finestra di dialogo Carico/Scarico piastra. Per selezionare i parametri di imaging, aprire la modalità Attivazione target nell'interfaccia di Cellomics BioApplications. Creare un nuovo protocollo per l'acquisizione dati a doppio canale compatibile con la colorazione nucleare e la sonda a fluorescenza DHE.Quindi, specificare gli obiettivi necessari per l'acquisizione dell'immagine, quindi scegliere l'ingrandimento appropriato. Ora specificate il tempo di esposizione, la piegatura della fotocamera e l'intervallo z-stack. Una volta impostati i parametri di imaging, identificare l'oggetto di tracciamento primario di interesse utilizzando i diversi algoritmi presenti nello strumento.Dopo aver segmentato l'oggetto identificato, convalidare l'oggetto principale in base ai parametri specifici di dimensione, forma e intensità univoci per ogni tipo di cella. Definisci la regione di interesse intorno all'oggetto e imposta un cerchio di 20 micrometri attorno ad esso. Fare clic sulla scheda Caratterizzazione della popolazione, quindi impostare il limite di scansione su 1000 celle per pozzetto con un minimo di 10 oggetti per campo.Ora fai clic su Avvia vista per scansionare ogni piastra a pozzetti. Quindi avvia il software di analisi della vista ed esporta i parametri dei dati quantitativi in formato CSV per un'ulteriore analisi. L'esposizione al perossido ha determinato un incremento statisticamente significativo della fluorescenza indotta dai ROS su tutte le linee cellulari in modo dose-dipendente.Con il menadione, le cellule JHH-4 hanno mostrato un aumento dose-dipendente dell'intensità fluorescente. Tuttavia, la differenza significativa nella fluorescenza è stata osservata solo a concentrazioni più elevate nelle altre due linee cellulari.

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 ...

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Bioingegneria