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

Neuartige ROS-Quantifizierung mit Dihydroethidium in Leberkrebszellen

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 im Rampenlicht: Hochdurchsatzmessung der intrazellulären ROS-Spiegel in hepatozellulären Linien

Hochdurchsatz-Screening-Bewertung der Erzeugung reaktiver Sauerstoffspezies (ROS) mit Dihydroethidium (DHE)-Fluoreszenzfarbstoff

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.

Dihydroethidium (DHE)-Färbung von Hepatokarzinomzellen zur Quantifizierung reaktiver Sauerstoffspezies (ROS)

Säen Sie zunächst 200 Mikroliter Volumen von 10.000 hepatozellulären Karzinomzellen in eine 96-Well-Platte mit DMEM und 10% FBS. Inkubieren Sie die Zellen über Nacht bei 37 Grad Celsius in einem befeuchteten Inkubator unter 5% Kohlendioxid. Ersetzen Sie am nächsten Tag die Nährmedien und behandeln Sie die Zellen mit unterschiedlichen Konzentrationen von Wasserstoffperoxid und Menadion.Lösen Sie fünf Milligramm DHE in einem Milliliter DMSO. Für die Arbeitslösung wird ein 100-Mikromol-Aliquot mit vorgewärmtem DMEM-Medium auf eine Konzentration von 10 Mikromol verdünnt. Wirbeln Sie die Arbeitsfarblösung 5 bis 10 Sekunden lang ein, um eine ordnungsgemäße Mischung zu gewährleisten.Entfernen Sie nun das Testmedium aus jeder Vertiefung, bevor Sie die Karzinomzellen aufstellen. Waschen Sie dann jede Mulde vorsichtig mit PBS. Pipettieren Sie 100 Mikroliter der Arbeitsfarblösung in jede Vertiefung.Inkubieren Sie die Platte 30 Minuten lang bei 37 Grad Celsius. Entfernen Sie das DHE-haltige Medium aus jeder Vertiefung. Verwenden Sie eine Mehrkanalpipette, um jede Vertiefung dreimal mit PBS zu waschen.Pipettieren Sie nun 200 Mikroliter PBS mit dem Kernfärbefarbstoff Hoechst 33342 in jede Vertiefung. Nach der Inkubation wird die Platte auf eine High-Content-Screening-Plattform überführt. Starten Sie die High-Content-Screening-Software Cellomics CX7.Kalibrieren Sie die Imaging-Plattform gemäß den Herstellerangaben. Öffnen Sie die für die Plattenmarke spezifischen Systemparameter in der Gerätedatenbank. Legen Sie anschließend die Platte mit den Proben vorsichtig auf den beheizten Tisch des Bildgebungssystems.Stellen Sie sicher, dass die Platte sicher positioniert und in der richtigen Ausrichtung ist. Drücken Sie dann vorsichtig auf die Mikroplatte, um sicherzustellen, dass sie flach auf dem Tisch anliegt. Sobald die Platte richtig positioniert ist, halten Sie die Strg-Taste gedrückt.Klicken Sie dann auf Strg und OK, um das Dialogfeld Plattenladen/-entladen zu öffnen. Um die Bildgebungsparameter auszuwählen, öffnen Sie den Target-Aktivierungsmodus in der Cellomics BioApplications-Oberfläche. Erstellen Sie ein neues Protokoll für die Zweikanal-Datenerfassung, das mit der Kernfärbung und der DHE-Fluoreszenzsonde kompatibel ist.Geben Sie als Nächstes die erforderlichen Objektive für die Bildaufnahme an und wählen Sie dann die entsprechende Vergrößerung. Geben Sie nun die Belichtungszeit, die Kamerabiegung und das Z-Stapel-Intervall an. Sobald die Bildgebungsparameter eingestellt sind, identifizieren Sie das primäre Tracking-Objekt von Interesse mithilfe der verschiedenen Algorithmen im Gerät.Nachdem Sie das identifizierte Objekt segmentiert haben, validieren Sie das primäre Objekt basierend auf den spezifischen Größen-, Form- und Intensitätsparametern, die für jeden Zellentyp eindeutig sind. Definieren Sie den interessierenden Bereich um das Objekt und legen Sie einen 20-Mikrometer-Kreis um das Objekt fest. Klicken Sie auf die Registerkarte Populationscharakterisierung und legen Sie dann das Scanlimit auf 1000 Zellen pro Well mit mindestens 10 Objekten pro Feld fest.Klicken Sie nun auf Launch View, um jede Well-Platte zu scannen. Starten Sie dann die Ansichtsanalysesoftware und exportieren Sie die quantitativen Datenparameter in einem CSV-Format für zusätzliche Analysen. Die Peroxidexposition führte zu einer statistisch signifikanten Zunahme der ROS-induzierten Fluoreszenz über alle Zelllinien in dosisabhängiger Weise.Mit Menadion zeigten die JHH-4-Zellen eine dosisabhängige Zunahme der Fluoreszenzintensität. Der signifikante Unterschied in der Fluoreszenz wurde jedoch nur bei höheren Konzentrationen in den beiden anderen Zelllinien beobachtet.

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 ...

Forschung

Biotechnik