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

Novel ROS Quantification Using Dihydroethidium in Liver Cancer Cells

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.

Author Spotlight: High-Throughput Measurement of Intracellular ROS Levels in Hepatocellular Lines

Screening med hög genomströmning av reaktiva syreradikaler (ROS) med hjälp av dihydroetid (DHE) fluorescensfärgämne

Dihydroetidium (DHE) färgning av hepatokarcinomceller för kvantifiering av reaktiva syrearter (ROS)

Till att börja med, så 200 mikroliter volym av 10 000 hepatocellulära karcinomceller i en 96-brunnsplatta med DMEM och 10%FBS. Inkubera cellerna över natten vid 37 grader Celsius i en fuktad inkubator under 5 % koldioxid. Nästa dag byter du ut odlingsmediet och behandlar cellerna med varierande koncentrationer av väteperoxid och menadion.Lös upp fem milligram DHE i en milliliter DMSO. För arbetslösningen, späd en alikvot på 100 mikromol med förvärmda DMEM-medier till en koncentration av 10 mikromol. Virvla arbetsfärglösningen i 5 till 10 sekunder för att säkerställa korrekt blandning.Ta nu bort testmediet från varje brunn innan du ställer cancercellerna upp. Tvätta sedan försiktigt varje väl med PBS. Pipettera 100 mikroliter av arbetsfärgslösningen i varje brunn.Inkubera plattan vid 37 grader Celsius i 30 minuter. Ta bort DHE-innehållande media från varje brunn. Använd en flerkanalspipett för att tvätta varje brunn tre gånger med PBS.Pipettera nu 200 mikroliter PBS som innehåller Hoechst 33342 kärnfärgningsfärg i varje brunn. Efter inkubationen överförs plattan till en screeningplattform med högt innehåll. Starta Cellomics CX7 programvara för screening med högt innehåll.Kalibrera bildplattformen enligt tillverkarens specifikationer. Öppna de systemparametrar som är specifika för plattmärket i instrumentdatabasen. Placera sedan försiktigt plattan med proverna på den uppvärmda stage av bildsystemet.Se till att plattan är säkert placerad och i rätt riktning. Tryck sedan försiktigt ner mikroplattan för att säkerställa att den vilar platt mot stage. När plattan är korrekt placerad, tryck och håll ned kontrollknappen.Klicka sedan på Kontroll och OK för att öppna dialogrutan Plåtlastning/lossning. För att välja bildparametrar, öppna målaktiveringsläget i Cellomics BioApplications-gränssnittet. Skapa ett nytt protokoll för datainsamling med dubbla kanaler som är kompatibelt med kärnfärgning och DHE-fluorescenssond.Ange sedan de mål som krävs för bildförvärv och välj sedan lämplig förstoring. Ange nu exponeringstid, kameraböjning och z-stackintervall. När bildparametrarna har ställts in, identifiera det primära spårningsobjektet av intresse med hjälp av de olika algoritmerna i instrumentet.När du har segmenterat det identifierade objektet validerar du det primära objektet baserat på de specifika parametrarna för storlek, form och intensitet som är unika för varje celltyp. Definiera intresseområdet runt objektet och ställ en cirkel på 20 mikrometer runt det. Klicka på fliken Populationskarakterisering och ställ sedan in skanningsgränsen till 1000 celler per brunn med minst 10 objekt per fält.Klicka nu på Launch View för att skanna varje brunnsplatta. Starta sedan programvaran för vyanalys och exportera de kvantitativa dataparametrarna i CSV-format för ytterligare analys. Peroxidexponering resulterade i en statistiskt signifikant ökning av ROS-inducerad fluorescens över alla cellinjer på ett dosberoende sätt.Med menadion uppvisade JHH-4-cellerna en dosberoende ökning av fluorescerande intensitet. Den signifikanta skillnaden i fluorescens observerades dock endast vid högre koncentrationer i de andra två cellinjerna.

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 ...