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

Ny ROS-kvantificering ved hjælp af dihydroethidium i leverkræftceller

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.

Forfatter Spotlight: Højkapacitetsmåling af intracellulære ROS-niveauer i hepatocellulære linjer

Vurdering af screening med høj kapacitet af generering af reaktive iltarter (ROS) ved hjælp af dihydroethidium (DHE) fluorescensfarvestof

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) farvning af hepatocarcinomceller til kvantificering af reaktive iltarter (ROS)

Til at begynde med, frø 200 mikroliter volumen af 10.000 hepatocellulære carcinomceller i en 96 brøndplade med DMEM og 10%FBS. Inkuber cellerne natten over ved 37 grader Celsius i en befugtet inkubator under 5% kuldioxid. Den næste dag skal du udskifte kulturmediet og behandle cellerne med varierende koncentrationer af hydrogenperoxid og menadion.Opløs fem milligram DHE i en milliliter DMSO. Til arbejdsløsningen fortyndes en 100 mikromol alikvote med forvarmede DMEM-medier til en koncentration på 10 mikromol. Vortex arbejdsfarvestofopløsningen i 5 til 10 sekunder for at sikre korrekt blanding.Fjern nu testmediet fra hver brønd, før du står karcinomcellerne. Derefter vaskes hver brønd forsigtigt med PBS. Pipette 100 mikroliter af arbejdsfarvestofopløsningen i hver brønd.Inkuber pladen ved 37 grader Celsius i 30 minutter. Fjern DHE-holdige medier fra hver brønd. Brug en flerkanalpipette til at vaske hvert hul tre gange med PBS.Der pipetteres nu 200 mikroliter PBS indeholdende Hoechst 33342 nukleærfarvningsfarvestof i hvert hul. Efter inkubation overføres pladen til en screeningsplatform med højt indhold. Start Cellomics CX7 screeningssoftware med højt indhold.Kalibrer billedplatformen i henhold til producentens specifikationer. Åbn de systemparametre, der er specifikke for plademærket, i instrumentdatabasen. Derefter placeres pladen forsigtigt med prøverne på billeddannelsessystemets opvarmede trin.Sørg for, at pladen er sikkert placeret og i den rigtige retning. Tryk derefter forsigtigt ned på mikropladen for at sikre, at den hviler fladt mod scenen. Når pladen er placeret korrekt, skal du trykke på ctrl-tasten og holde den nede.Klik derefter på Control og OK for at åbne dialogboksen Plate Load / Unload. For at vælge billeddannelsesparametrene skal du åbne målaktiveringstilstanden i grænsefladen Cellomics BioApplications. Opret en ny protokol til dobbeltkanals dataindsamling, der er kompatibel med nuklear farvning og DHE-fluorescenssonde.Angiv derefter de nødvendige mål for billedoptagelse, og vælg derefter den passende forstørrelse. Angiv nu eksponeringstid, kamerabøjning og z-stack-interval. Når billedparametrene er indstillet, skal du identificere det primære sporingsobjekt af interesse ved hjælp af de forskellige algoritmer i instrumentet.Når du har segmenteret det identificerede objekt, skal du validere det primære objekt baseret på de specifikke størrelses-, form- og intensitetsparametre, der er entydige for hver celletype. Definer interesseområdet omkring objektet, og indstil en cirkel på 20 mikrometer omkring det. Klik på fanen Populationskarakterisering, og indstil derefter scanningsgrænsen til 1000 celler pr. brønd med mindst 10 objekter pr. felt.Klik nu på Start visning for at scanne hver brøndplade. Start derefter visningsanalysesoftwaren, og eksporter de kvantitative dataparametre i et CSV-format til yderligere analyse. Peroxideksponering resulterede i en statistisk signifikant forøgelse af den ROS-inducerede fluorescens på tværs af alle cellelinjerne på en dosisafhængig måde.Med menadion viste JHH-4-cellerne en dosisafhængig stigning i fluorescerende intensitet. Den signifikante forskel i fluorescens blev imidlertid kun observeret ved højere koncentrationer i de to andre cellelinjer.

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 ...