RK and RRG were supported by a grant from the UNM Center for Metals in Biology and Medicine (CMBM) through NIH NIGMS grant P20 GM130422. RRG was supported by a pilot award from the NM-INSPIRES P30 grant 1P30ES032755. The imaging core support for the CX7 Cellomics instrument was provided through the AIM center cores funded by NIH grant P20GM121176.&#160;We would like to thank Dr. Sharina Desai and Dr. Li Chen for their invaluable assistance with technical issues related to the use of the CX7 Cellomics imaging platform.

1. Cell culture
<ol>
	<li>Seed the test cells (HepG2, HUH7, and JHH4 hepatocellular carcinoma cells) into a 96-well plate at a seeding density of 10,000 cells/well in a final seeding volume of 200 &#181;L per well.
	<ol>
		<li>Before culturing the HepG2 cells, coat the 96 plate wells with type IV collagen (50 &#181;g/mL) for 2 h duration at room temperature (RT). To avoid solidification of the stock collagen, place the stock solution into ice and subsequently, initiate the dilution process to the desir...

Novel ROS Quantification Using Dihydroethidium in Liver Cancer Cells

<ol>
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A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+chemistry+of+reactive+oxygen+species+(ros)+revisited:+Outlining+their+role+in+biological+macromolecules+(DNA,+Lipids+and+Proteins)+and+induced+pathologies.">The chemistry of reactive oxygen species (ros) revisited: Outlining their role in biological macromolecules (DNA, Lipids and Proteins) and induced pathologies.</a> Int J Mol Sci. 22 (9), 4642 (2021).</li><li>Magnani, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+and+mechanisms+of+ROS+generation+by+NADPH+oxidases.">Structure and mechanisms of ROS generation by NADPH oxidases.</a> Curr Opin Struct Biol. 59, 91-97 (2019).</li><li>Collin, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+basis+of+reactive+oxygen+species+reactivity+and+involvement+in+neurodegenerative+diseases.">Chemical basis of reactive oxygen species reactivity and involvement in neurodegenerative diseases.</a> Int J Mol Sci. 20 (10), 2407 (2019).</li><li>Uttara, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxidative+stress+and+neurodegenerative+diseases:+a+review+of+upstream+and+downstream+antioxidant+therapeutic+options.">Oxidative stress and neurodegenerative diseases: a review of upstream and downstream antioxidant therapeutic options.</a> Curr Neuropharmacol. 7 (1), 65-74 (2009).</li><li>Dikalov, S. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electron+paramagnetic+resonance+measurements+of+reactive+oxygen+species+by+cyclic+hydroxylamine+spin+probes.">Electron paramagnetic resonance measurements of reactive oxygen species by cyclic hydroxylamine spin probes.</a> Antioxid Redox Signal. 28 (15), 1433-1443 (2018).</li><li>Wang, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+reactive+oxygen+species+(ROS)+and+mitochondrial+ROS+in+AMPK+knockout+mice+blood+vessels.">Measurement of reactive oxygen species (ROS) and mitochondrial ROS in AMPK knockout mice blood vessels.</a> Methods Mol Biol. 1732, 507-517 (2018).</li><li>Griendling, K. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+reactive+oxygen+species,+reactive+nitrogen+species,+and+redox-dependent+signaling+in+the+cardiovascular+system:+a+scientific+statement+from+the+American+Heart+Association.">Measurement of reactive oxygen species, reactive nitrogen species, and redox-dependent signaling in the cardiovascular system: a scientific statement from the American Heart Association.</a> Circ Res. 119 (5), e39-e75 (2016).</li><li>Kalyanaraman, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+reactive+oxygen+and+nitrogen+species+with+fluorescent+probes:+challenges+and+limitations.">Measuring reactive oxygen and nitrogen species with fluorescent probes: challenges and limitations.</a> Free Radic Biol Med. 52 (1), 1-6 (2012).</li><li>Gomes, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+probes+used+for+detection+of+reactive+oxygen+species.">Fluorescence probes used for detection of reactive oxygen species.</a> J Biochem Biophys Methods. 65 (2-3), 45-80 (2005).</li><li>Halliwell, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Free radicals in biology and medicine. , (2015).</li><li>Cho, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence-based+detection+and+quantification+of+features+of+cellular+senescence.">Fluorescence-based detection and quantification of features of cellular senescence.</a> Methods Cell Biol. 103, 149-188 (2011).</li><li>Dikalov, S. 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M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+fluorescent+imaging+of+superoxide+in+vivo+using+ethidium-based+probes.">Selective fluorescent imaging of superoxide in vivo using ethidium-based probes.</a> Proc Natl Acad Sci U.S.A. 103 (41), 15038-15043 (2006).</li><li>Zielonka, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+2-hydroxyethidium+in+cellular+systems:+a+unique+marker+product+of+superoxide+and+hydroethidine.">Detection of 2-hydroxyethidium in cellular systems: a unique marker product of superoxide and hydroethidine.</a> Nat Protoc. 3 (1), 8-21 (2008).</li><li>Nazarewicz, R. 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P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guidelines+for+measuring+reactive+oxygen+species+and+oxidative+damage+in+cells+and+in+vivo.">Guidelines for measuring reactive oxygen species and oxidative damage in cells and in vivo.</a> Nat Metab. 4 (6), 651-662 (2022).</li><li>Ameziane-El-Hassani, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+reactive+oxygen+species+in+cells+undergoing+oncogene-induced+senescence.">Detection of reactive oxygen species in cells undergoing oncogene-induced senescence.</a> Oncogene-Induced Senescence: Methods Protoc. 1534, 139-145 (2017).</li><li>Abraham, , et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+content+screening+applied+to+large-scale+cell+biology.">High content screening applied to large-scale cell biology.</a> Trends Biotechnol. 22 (1), 15-22 (2004).</li><li>Criddle, D. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Menadione-induced+reactive+oxygen+species+generation+via+redox+cycling+promotes+apoptosis+of+murine+pancreatic+acinar+cells.">Menadione-induced reactive oxygen species generation via redox cycling promotes apoptosis of murine pancreatic acinar cells.</a> J Biol Chem. 281 (52), 40485-40492 (2006).</li><li>Goffart, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+type+and+source+of+reactive+oxygen+species+influences+the+outcome+of+oxidative+stress+in+cultured+cells.">The type and source of reactive oxygen species influences the outcome of oxidative stress in cultured cells.</a> Cells. 10 (5), 1075 (2021).</li><li>Kurutas, E. 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The authors declare that they have no competing interests.

In this study, a protocol to assess superoxide-driven intracellular reactive oxygen species (ROS) production using dihydroethidium (DHE) fluorescence dye was established on a high-content screening system. A majority of the current protocols available in the literature use the DCFH-DA as a fluorescence imaging probe to quantify ROS species. However, multiple studies have shown the DCFH-DA is not an ideal probe for the measurement of intracellular ROS. Various reasons postulated for the unsuitability of DCFH-DA as a probe...

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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1.5 mL centrifuge tubes&#160;</td>
			<td>VWR&#160;</td>
			<td>20170-038&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>96- well plate&#160;</td>
			<td>Corning Costar&#160;</td>
			<td>07-200-90&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Cellomics Cx7</td>
			<td>ThermoFisher&#160;</td>
			<td>HCSDCX7LEDPRO</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagen&#160;</td>
			<td>Advanced Biomatrix&#160;&#160;</td>
			<td>5056&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>DHE (Dihydroethidium)&#160;</td>
			<td>ThermoFisher&#160;</td>
			<td>D1168&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM&#160;</td>
			<td>Sigma&#160;&#160;</td>
			<td>6046&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS&#160;</td>
			<td>VWR&#160;</td>
			<td>97068-085&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>GraphPad Prism</td>
			<td>GraphPad</td>
			<td>Version 6.0</td>
			<td></td>
		</tr>
		<tr>
			<td>HepG2 cell line</td>
			<td>ATCC</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Hoechst&#160;</td>
			<td>ThermoFisher&#160;</td>
			<td>33342&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>HUH7 cell line</td>
			<td>ATCC</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrogen Peroxide&#160;</td>
			<td>Sigma&#160;</td>
			<td>88597&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>JHH4 cell line</td>
			<td>ATCC</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Menadione&#160;</td>
			<td>Sigma&#160;</td>
			<td>M5625&#160;</td>
			<td></td>
		</tr>
	</tbody>
</table>

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 production plays a central role in the spatial and temporal modulation of normal cellular functions such as proliferation, signaling, apoptosis, and senescence. In contrast, chronic ROS overproduction is responsible for a wide spectrum of diseases, such as cancer, cardiovascular disease, and diabetes, among others. Quantifying ROS levels in an accurate and reproducible manner is thus essential to understanding normal cellular functionality. Fluorescence imaging-based methods to characterize intra-cellular ROS species is a common approach. Many of the imaging ROS protocols in the literature use 2&#39;-7&#39;-dichlorodihydrofluorescein diacetate (DCFH-DA) dye. However, this dye suffers from significant limitations in its usage and interpretability. The current protocol demonstrates the use of a dihydroethidium (DHE) fluorescent probe as an alternative method to quantify total ROS production in a high-throughput setting. The high throughput imaging platform, CX7 Cellomics, was used to measure and quantify the ROS production. This study was conducted in three hepatocellular cancer cell lines - HepG2, JHH4, and HUH-7. This protocol provides an in-depth description of the various procedures involved in the assessment of ROS within the cells, including - preparation of DHE solution, incubation of cells with DHE solution, and measurement of DHE intensity necessary to characterize the ROS production. This protocol demonstrates that DHE fluorescent dye is a robust and reproducible choice to characterize intracellular ROS production in a high-throughput manner. High throughput approaches to measure ROS production are likely to be helpful in a variety of studies, such as toxicology, drug screening, and cancer biology.

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

High Throughput Screening Assessment of Reactive Oxygen Species (ROS) Generation using Dihydroethidium (DHE) Fluorescence Dye

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) is a superoxide-responsive fluorescence dye that provides specific information regarding the intracellular ROS states. DHE dye intrinsically emits blue fluorescence in the cytoplasm. However, upon interaction with superoxide radicals, it is transformed into 2-hydroxyethidium, which emits fluorescence in the red wavelengths (&#62;550 nm) (Figure 1). DHE dye is easily transported into the cells and the nucleus. The fluorescence emitted can be visualized with a fluorescenc...

Watch this Scientific Journal Video about High Throughput Screening Assessment of Reactive Oxygen Species ROS Generation using Dihydroethidium DHE Fluorescence Dye at JoVE.com

This protocol describes a novel method to quantify intracellular reactive oxygen species (ROS) using dihydroethidium (DHE) as a fluorescence dye probe using a high-throughput screening approach. The protocol describes the methods for quantitative assessment of intracellular reactive oxygen species (ROS) in the three different hepatocellular carcinoma cell lines.

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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Dihydroethidium &#40;DHE&#41; Staining of Hepatocarcinoma Cells for the Quantification of Reactive Oxygen Species &#40;ROS&#41;

2.6K Views.  University of New Mexico.  This protocol describes a novel method to quantify intracellular reactive oxygen species (ROS) using dihydroethidium (DHE) as a fluorescence dye probe using a high-throughput screening approach. The protocol describes the methods for quantitative assessment of intracellular reactive oxygen species (ROS) in the three different hepatocellular carcinoma cell lines.

Novel ROS Quantification Using Dihydroethidium in Liver Cancer Cells

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