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

Nieuwe ROS-kwantificering met behulp van dihydroethidium in leverkankercellen

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.

Auteur in de schijnwerpers: High-throughput meting van intracellulaire ROS-niveaus in hepatocellulaire lijnen

High Throughput Screening Assessment van het genereren van reactieve zuurstofsoorten (ROS) met behulp van dihydroethidium (DHE) fluorescentiekleurstof

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) kleuring van hepatocarcinoomcellen voor de kwantificering van reactieve zuurstofsoorten (ROS)

Zaai om te beginnen 200 microliter volume van 10.000 hepatocellulaire carcinoomcellen in een plaat met 96 putjes met DMEM en 10% FBS. Incubeer de cellen 's nachts bij 37 graden Celsius in een bevochtigde incubator onder 5% koolstofdioxide. Vervang de volgende dag de kweekmedia en behandel de cellen met verschillende concentraties waterstofperoxide en menadion.Los vijf milligram DHE op in één milliliter DMSO. Verdun voor de werkoplossing een aliquot van 100 micromol met voorverwarmde DMEM-media tot een concentratie van 10 micromol. Draai de werkkleurstofoplossing gedurende 5 tot 10 seconden om een goede menging te garanderen.Verwijder nu de testmedia uit elk putje voordat u de carcinoomcellen laat staan. Was vervolgens elk putje voorzichtig met PBS. Pipetteer 100 microliter van de werkende kleurstofoplossing in elk putje.Incubeer de plaat 30 minuten op 37 graden Celsius. Verwijder de DHE-bevattende media uit elk putje. Gebruik een multikanaalspipet om elk putje drie keer met PBS te wassen.Pipetteer nu 200 microliter PBS met Hoechst 33342 nucleaire kleurstof in elk putje. Breng de plaat na incubatie over naar een zeefplatform met een hoog gehalte. Start de Cellomics CX7 high-content screeningsoftware.Kalibreer het beeldvormingsplatform volgens de specificaties van de fabrikant. Open de systeemparameters die specifiek zijn voor het plaatmerk in de instrumentendatabase. Plaats vervolgens de plaat met de monsters voorzichtig op de verwarmde tafel van het beeldvormingssysteem.Zorg ervoor dat de plaat stevig en in de juiste richting is geplaatst. Druk vervolgens voorzichtig op de microplaat om ervoor te zorgen dat deze plat tegen de tafel rust. Zodra de plaat correct is geplaatst, houdt u de Control-toets ingedrukt.Klik vervolgens op Control en OK om het dialoogvenster Plate Load/Unload te openen. Om de beeldvormingsparameters te selecteren, opent u de Target Activeringsmodus in de Cellomics BioApplications-interface. Creëer een nieuw protocol voor tweekanaals data-acquisitie dat compatibel is met nucleaire kleuring en DHE-fluorescentiesonde.Specificeer vervolgens de vereiste doelstellingen voor beeldacquisitie en kies vervolgens de juiste vergroting. Geef nu de belichtingstijd, de buiging van de camera en het z-stack-interval op. Zodra de beeldvormingsparameters zijn ingesteld, identificeert u het primaire volgobject van belang met behulp van de verschillende algoritmen in het instrument.Nadat u het geïdentificeerde object hebt gesegmenteerd, valideert u het primaire object op basis van de specifieke grootte-, vorm- en intensiteitsparameters die uniek zijn voor elk celtype. Definieer het interessegebied rond het object en plaats er een cirkel van 20 micrometer omheen. Klik op het tabblad Populatiekarakterisering en stel vervolgens de scanlimiet in op 1000 cellen per put met een minimum van 10 objecten per veld.Klik nu op Launch View om elke putplaat te scannen. Start vervolgens de weergaveanalysesoftware en exporteer de kwantitatieve gegevensparameters in een CSV-indeling voor aanvullende analyse. Blootstelling aan peroxide resulteerde in een statistisch significante toename van de ROS-geïnduceerde fluorescentie over alle cellijnen op een dosisafhankelijke manier.Met menadion vertoonden de JHH-4-cellen een dosisafhankelijke toename van de fluorescerende intensiteit. Het significante verschil in fluorescentie werd echter alleen waargenomen bij hogere concentraties in de andere twee cellijnen.

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 ...