Name: detection of total reactive oxygen species in adherent cells by 2',7'-dichlorodihydrofluorescein diacetate staining
Uploaded: 2020-06-23T13:00:00
Description: Watch this Scientific Journal Video about Detection of Total Reactive Oxygen Species in Adherent Cells by 2',7'-Dichlorodihydrofluorescein Diacetate Staining at JoVE.com

This work was supported in part by the National Institutes of Health (K01DK114390), a Research Scholar Grant from the American Cancer Society (RSG-18-050-01-NEC), a Research Pilot Project Grant from University of New Mexico Environmental Health Signature Program and Superfund (P42 ES025589), a Shared Resources Pilot Project Award and a Research Program Support Pilot Project Award from UNM comprehensive cancer center (P30CA118100), and a new investigator award from the Dedicated Health Research Funds at the University of New Mexico School of Medicine.

1. Cell seeding
<ol>
	<li>Seed 2 x 105 HCT116 colorectal cancer cells per well in a 24-well plate and maintain the cells in Dulbecco&#39;s modified Eagle medium (DMEM) overnight at 37 &#176;C.</li>
	<li>Replace the culture medium with or without 100 &#181;M ferrous sulfate (FS) or 10 &#181;M doxorubicin (DOX) containing medium and incubate for 24 h.</li>
</ol>
2. Preparation of the DCFH-DA solution
<ol>
	<li>Dissolve 4.85 mg of DCFH-DA in 1 mL of dimethyl sulfoxide (DMSO) to make a ...

<ol>
	<li>Birben, E., 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+antioxidant+defense.">Oxidative stress and antioxidant defense.</a> World Allergy Organization Journal. 5 (1), 9-19 (2012).</li><li>Kim, G. H., 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+Role+of+Oxidative+Stress+in+Neurodegenerative+Diseases.">The Role of Oxidative Stress in Neurodegenerative Diseases.</a> Experimental Neurobiology. 24 (4), 325-340 (2015).</li><li>Sullivan, L. B., Chandel, N. 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H., 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=Sotetsuflavone+inhibits+proliferation+and+induces+apoptosis+of+A549+cells+through+ROS-mediated+mitochondrial-dependent+pathway.">Sotetsuflavone inhibits proliferation and induces apoptosis of A549 cells through ROS-mediated mitochondrial-dependent pathway.</a> BMC Complementary and Alternative Medicine. 18, 235 (2018).</li><li>Kruger, N. J., Walker, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Bradford+Method+For+Protein+Quantitation.">The Bradford Method For Protein Quantitation.</a> The Protein Protocols Handbook. , 17-24 (2009).</li><li>Tetz, L. 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The experimental protocol described here is easily reproducible to measure cellular total ROS. The critical steps include making DCFH-DA solution fresh and avoiding light exposure, minimizing cell status disturbance and extensive PBS washing right before taking images. For the preparation of DCFH-DA working solution, the stock solution should be added into pre-warmed DMEM right before adding into the 24 well plate. The reason is that old solutions that generate high background fluorescence or light exposure will lead to ...

Three major reactive oxygen species (ROS) produced by cellular metabolism that are of physiological meaning are superoxide anion, hydroxyl radical, and hydrogen peroxide1. At low concentrations, they participate in physiological cell processes, but at high concentrations they have adverse effects on cell signaling pathways1. Our body has developed antioxidant systems, which are effective against excessive ROS. However, oxidative stress can occur when ROS overwhelm the detoxifying ability of our body, which contributes to many pathological conditions, including inflammation, cancer, and neurodegenerative disease2,3,4. The purpose of this method is to determine total cellular ROS in adherent cells using 2&#39;,7&#39;-dichlorodihydrofluorescein diacetate (DCFH-DA) staining. The rationale is that oxidation of DCFH-DA to 2&#8217;-7&#8217;dichlorofluorescein (DCF) has been used extensively for total ROS detection including hydroxyl radicals (&#8226;OH) and nitrogen dioxide (&#8226;NO2). Mechanistically, DCFH-DA is taken up by cells where cellular esterase cleaves off the acetyl groups, resulting in DCFH. Oxidation of DCFH by ROS converts the molecule to DCF, which emits green fluorescence at an excitation wavelength of 485 nm and an emission wavelength of 530 nm. Compared with detection of fluorescence with flow cytometry and other alternative methods5, advantages of this method using a fluorescence microscope and a plate reader are that it produces clearly visible fluorescent images, and is easy to perform, efficient and cost-effective. This method has been widely used to detect cellular ROS for studying various conditions6,7,8. This protocol is used for detecting total ROS in adherent cells. Using this method to detect ROS in suspension cells may need some modifications.

<table border="0" cellpadding="0" cellspacing="0" style="width:900px;" width="899">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">2&#39;,7&#39;-Dichlorofluorescein diacetate</td>
			<td style="width:229px;">Cayman Chemical, Ann Arbor, MI</td>
			<td style="width:93px;">20656</td>
			<td style="width:300px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Doxorubicin hydrochloride</td>
			<td style="width:229px;">TCI America, Portland, OR</td>
			<td style="width:93px;">D4193-25MG</td>
			<td style="width:300px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">Dulbecco&#39;s Modified Eagle Medium</td>
			<td>Corning, Corning, NY</td>
			<td>45000-304</td>
			<td style="width:300px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">Ferrous Sulfate Heptahydrate</td>
			<td style="width:229px;">VWR, Radnor, PA</td>
			<td style="width:93px;">97061-542</td>
			<td style="width:300px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Invitrogen EVOS FL Auto Imaging System</td>
			<td style="width:229px;">Thermo Fisher Scientific Waltham, MA</td>
			<td>AMAFD1000</td>
			<td>or any other fluorescence microscope</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Protein assay Bradford solution</td>
			<td>Bio-Rad, Hercules, CA</td>
			<td style="width:93px;">5000001</td>
			<td style="width:300px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SpectraMax M2 Microplate Reader</td>
			<td>Molecular Devices, Radnor, PA</td>
			<td>89429-532</td>
			<td>or any other fluorescence microplate reader</td>
		</tr>
	</tbody>
</table>

detection of total reactive oxygen species in adherent cells by 2',7'-dichlorodihydrofluorescein diacetate staining

Oxidative stress is an important event under both physiological and pathological conditions. In this study, we demonstrate how to quantify oxidative stress by measuring total reactive oxygen species (ROS) using 2&#39;,7&#39;-dichlorodihydrofluorescein diacetate (DCFH-DA) staining in colorectal cancer cell lines as an example. This protocol describes detailed steps including preparation of DCFH-DA solution, incubation of cells with DCFH-DA solution, and measurement of normalized intensity. DCFH-DA staining is a simple and cost-effective way to detect ROS in cells. It can be used to measure ROS generation after chemical treatment or genetic modifications. Therefore, it is useful for determining cellular oxidative stress upon environment stress, providing clues to mechanistic studies.

HCT116 colorectal cancer cells were treated with 100 &#181;M FS or 10 &#181;M DOX to induce oxidative stress7. As shown in Figure 1, green fluorescence was dramatically increased by both FS and DOX as expected. To quantify the relative intensity change, the cells were lysed after taking images and normalized with protein concentrations. The quantified fluorescence intensity was significantly increased by FS or DOX in HCT116 cells.
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Watch this Scientific Journal Video about Detection of Total Reactive Oxygen Species in Adherent Cells by 2',7'-Dichlorodihydrofluorescein Diacetate Staining at JoVE.com

Detection of Total Reactive Oxygen Species in Adherent Cells by 2&#039;,7&#039;-Dichlorodihydrofluorescein Diacetate Staining

Here, we present a protocol to detect total cellular reactive oxygen species (ROS) using 2&#39;,7&#39;-dichlorodihydrofluorescein diacetate (DCFH-DA). This method can visualize cellular ROS localization in adherent cells with a fluorescence microscope and quantify ROS intensity with a fluorescence plate reader. This protocol is simple, efficient and cost-effective.

Detection of Total Reactive Oxygen Species in Adherent Cells by 2',7'-Dichlorodihydrofluorescein Diacetate Staining

Oxidative stress is an important event under both physiological and pathological conditions. In this study, we demonstrate how to quantify oxidative ...

This method can be used to analyze cellular reactive oxygen species in adherent cells with only a fluorescence microscope, and quantify reactive oxygen species intensity with fluorescence plate reader. This protocol is simple, efficient, and cost-effective. Start by seeding two times 10 to the fifth HCT116 colorectal cancer cells in DMEM into each well of a 24-well plate.Incubate the cells overnight at 37 degrees Celsius. On the next day, replace the culture medium with 100 micromolar ferrous sulfate, or 10 micromolar doxorubicin-containing medium, and incubate the plate for another 24 hours. Prepare the DCFH-DA solution by dissolving 4.85 milligrams of DCFH-DA in one milliliter of DMSO to make a 10 millimolar stock solution.Right before adding it to the wells, dilute the stock with pre-warmed DMEM to make a 10 micromolar working solution, and vortex it for 10 seconds. To stain the cells, remove the drug-containing medium, and wash them once with DMEM. Add 500 microliters of DCFH-DA working solution into each well, and incubate the plate at 37 degrees Celsius for 30 minutes.After the incubation, remove the DCFH-DA from the wells, and wash them twice with DMEM, and once with PBS. Then, add 500 microliters of PBS to each well. Use the green fluorescent protein channel on the fluorescence microscope to take representative images of the cells.Then remove the PBS, and add 200 microliters of radioimmunoprecipitation assay buffer to each well. Incubate the plate on ice for five minutes, and collect the cell lysates into 1.5 milliliter tubes. Centrifuge the tubes at 21, 130 times G for 10 minutes at four degrees Celsius.Then transfer 100 microliters of the supernatant to a black 96-well plate. Use a microplate reader at an excitation wavelength of 485 nanometers, and an emission wavelength of 530 nanometers, to measure the fluorescence in each well. Next, measure the protein concentration with the Bradford assay by transferring one microliter of the supernatant to a clear 96-well plate with 100 microliters of protein assay solution.Use the protein concentrations to normalize the fluorescence intensities. HCT116 colorectal cancer cells were treated with 100 micromolar ferrous sulfate, or 10 micromolar doxorubicin, to induce oxidative stress. As expected, green fluorescence was dramatically increased by both treatments.To quantify the relative intensity changes, the cells were lysed and normalized with protein concentrations. The relative fluorescence intensity was significantly increased by ferrous sulfate or doxorubicin. When performing this procedure, it is important to make DCFH-DA solution fresh and avoid light exposure, as well as to minimize cell status disturbance, and to perform extensive PBS wash right before taking image.In addition to detection of the total ROS by this protocol, specifically ROS such as peroxide can be performed.

detection-total-reactive-oxygen-species-adherent-cells-2-7

Research

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Biochemistry

Quantitative and Qualitative Method for Sphingomyelin by LC-MS Using Two Stable Isotopically Labeled Sphingomyelin Species

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Here, we present a protocol to quantify and qualify each sphingomyelin species using multiple reaction monitoring and MS/MS/MS mode, respectively.

We present a method of analyzing sphingomyelin (SM) qualitatively and quantitatively by liquid chromatography-electrospray Ionization-tandem mass ...

Dissipative Microgravimetry to Study the Binding Dynamics of the Phospholipid Binding Protein Annexin A2 to Solid-supported Lipid Bilayers Using a Quartz Resonator

The dissipative quartz crystal microbalance technique is a simple and label-free approach to measure simultaneously the mass uptake and viscoelastic properties of the absorbed/immobilized mass on sensor surfaces, allowing the measurements of the interaction of proteins with solid-supported surfaces, such as lipid bilayers, in real-time and with a high sensitivity. Annexins are a highly conserved group of phospholipid-binding proteins that interact reversibly with the negatively charged headgroups via the coordination of calcium ions. Here, we describe a protocol that was employed to quantitatively analyze the binding of annexin A2 (AnxA2) to planar lipid bilayers prepared on the surface of a quartz sensor. This protocol is optimized to obtain robust and reproducible data and includes a detailed step-by-step description. The method can be applied to other membrane-binding proteins and bilayer compositions.

Here, we present an experimental protocol that can be employed to determine the binding affinities and mode of interaction of label-free phospholipid-binding protein annexin A2 with immobilized solid-supported bilayers (SLB) by simultaneously measuring the mass uptake and the viscoelastic properties of the protein annexin A2.

The dissipative quartz crystal microbalance technique is a simple and label-free approach to measure simultaneously the mass uptake and viscoelastic ...

Measuring and Interpreting Oxygen Consumption Rates in Whole Fly Head Segments

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Measuring alterations in metabolic rates is central to understanding the progression of various diseases and aging. Here, we present a novel technique to measure whole head oxygen consumption that more closely resembles the physiological state and may aid in revealing novel drugs that modify mitochondrial activity.

Regulated metabolic activity is essential for the normal functioning of living cells. Indeed, altered metabolic activity is causally linked with the ...

Fluorescent Silver Staining of Proteins in Polyacrylamide Gels

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Here, we describe a detailed protocol outlining a new fluorescent staining technique for total protein detection in polyacrylamide gels. The protocol utilizes a silver ion-specific fluorescence turn-on probe, which detects Ag+-protein complexes, and eliminates certain limitations of traditional chromogenic silver stains.

Silver staining is a colorimetric technique widely used to visualize protein bands in polyacrylamide gels following sodium dodecyl sulfate-polyacrylamide ...

Using High Content Imaging to Quantify Target Engagement in Adherent Cells

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Measurements of drug target engagement are central to effective drug development and chemical probe validation. Here, we detail a protocol for measuring drug-target engagement using high content imaging in a microplate-compatible adaption of the cellular thermal shift assay (CETSA).

Quantitating the interaction of small molecules with their intended protein target is critical for drug development, target validation and chemical probe ...

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Here we describe a protocol for a general pulse-chase method that allows the kinetic analysis of folding, transport, and degradation of proteins to be followed in live cells.

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Detection of Protein Ubiquitination Sites by Peptide Enrichment and Mass Spectrometry

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We present a method for the purification, detection, and identification of diGly peptides that originate from ubiquitinated proteins from complex biological samples. The presented method is reproducible, robust, and outperforms published methods with respect to the level of depth of the ubiquitinome analysis.

The posttranslational modification of proteins by the small protein ubiquitin is involved in many cellular events. After tryptic digestion of ...

Study on the Metabolism of Six Systemic Insecticides in a Newly Established Cell Suspension Culture Derived from Tea (Camellia Sinensis L.) Leaves

A platform for studying insecticide metabolism using in vitro tissues of tea plant was developed. Leaves from sterile tea plantlets were induced to form loose callus on Murashige and Skoog (MS) basal media with the plant hormones 2,4-dichlorophenoxyacetic acid (2,4-D, 1.0 mg L-1) and kinetin (KT, 0.1 mg L-1). Callus formed after 3 or 4 rounds of subculturing, each lasting 28 days. Loose callus (about 3 g) was then inoculated into B5 liquid media containing the same plant hormones and was cultured in a shaking incubator (120 rpm) in the dark at 25 &#177; 1 &#176;C. After 3&#8722;4 subcultures, a cell suspension derived from tea leaf was established at a subculture ratio ranging between 1:1 and 1:2 (suspension mother liquid: fresh medium). Using this platform, six insecticides (5 &#181;g mL-1 each thiamethoxam, imidacloprid, acetamiprid, imidaclothiz, dimethoate, and omethoate) were added into the tea leaf-derived cell suspension culture. The metabolism of the insecticides was tracked using liquid chromatography and gas chromatography. To validate the usefulness of the tea cell suspension culture, the metabolites of thiamethoxan and dimethoate present in treated cell cultures and intact plants were compared using mass spectrometry. In treated tea cell cultures, seven metabolites of thiamethoxan and two metabolites of dimethoate were found, while in treated intact plants, only two metabolites of thiamethoxam and one of dimethoate were found. The use of a cell suspension simplified the metabolic analysis compared to the use of intact tea plants, especially for a difficult matrix such as tea.

Study on the Metabolism of Six Systemic Insecticides in a Newly Established Cell Suspension Culture Derived from Tea Camellia Sinensis L. Leaves

This work presents a protocol for establishing a cell suspension culture derived from tea (Camellia sinensis L.) leaves that can be used to study the metabolism of external compounds that can be taken up by the whole plant, such as insecticides.

A platform for studying insecticide metabolism using in vitro tissues of tea plant was developed. Leaves from sterile tea plantlets were induced to form ...

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Visualizing Actin and Microtubule Coupling Dynamics In Vitro by Total Internal Reflection Fluorescence TIRF Microscopy

This protocol is a guide for visualizing dynamic actin and microtubules using an in vitro total internal fluorescence (TIRF) microscopy assay.

Traditionally, the actin and microtubule cytoskeletons have been studied as separate entities, restricted to specific cellular regions or processes, and ...

Stable Isotope Tracing &amp; LC-MS Analysis to Measure DHODH and SDH Activities

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The flow of electrons in the mitochondrial electron transport chain (ETC) supports multifaceted biosynthetic, bioenergetic, and signaling functions in mammalian cells. As oxygen (O2) is the most ubiquitous terminal electron acceptor for the mammalian ETC, the O2 consumption rate is frequently used as a proxy for mitochondrial function. However, emerging research demonstrates that this parameter is not always indicative of mitochondrial function, as fumarate can be employed as an alternative electron acceptor to sustain mitochondrial functions in hypoxia. This article compiles a series of protocols that allow researchers to measure mitochondrial function independently of the O2 consumption rate. These assays are particularly useful when studying mitochondrial function in hypoxic environments. Specifically, we describe methods to measure mitochondrial ATP production, de novo pyrimidine biosynthesis, NADH oxidation by complex I, and superoxide production. In combination with classical respirometry experiments, these orthogonal and economical assays will provide researchers with a more comprehensive assessment of mitochondrial function in their system of interest.

Author Spotlight: Oxygen-Independent Assays to Measure Mitochondrial Function in Mammals  

Here, we present a compilation of assays to directly measure mitochondrial function in mammalian cells independently of their ability to consume molecular oxygen.

The flow of electrons in the mitochondrial electron transport chain (ETC) supports multifaceted biosynthetic, bioenergetic, and signaling functions in ...