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. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+reactive+oxygen+species+and+cancer.">Mitochondrial reactive oxygen species and cancer.</a> Cancer &amp; Metabolism. 2, 17 (2014).</li><li>Formentini, L., 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=Mitochondrial+ROS+Production+Protects+the+Intestine+from+Inflammation+through+Functional+M2+Macrophage+Polarization.">Mitochondrial ROS Production Protects the Intestine from Inflammation through Functional M2 Macrophage Polarization.</a> Cell Reports. 19 (6), 1202-1213 (2017).</li><li>Rakotoarisoa, 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=Curcumin-+and+Fish+Oil-Loaded+Spongosome+and+Cubosome+Nanoparticles+with+Neuroprotective+Potential+against+H2O2-Induced+Oxidative+Stress+in+Differentiated+Human+SH-SY5Y+Cells.">Curcumin- and Fish Oil-Loaded Spongosome and Cubosome Nanoparticles with Neuroprotective Potential against H2O2-Induced Oxidative Stress in Differentiated Human SH-SY5Y Cells.</a> ACS Omega. 4 (2), 3061-3073 (2019).</li><li>Mateen, 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=Increased+Reactive+Oxygen+Species+Formation+and+Oxidative+Stress+in+Rheumatoid+Arthritis.">Increased Reactive Oxygen Species Formation and Oxidative Stress in Rheumatoid Arthritis.</a> PLoS One. 11 (4), (2016).</li><li>Kim, 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+interaction+of+Hemin+and+Sestrin2+modulates+oxidative+stress+and+colon+tumor+growth.">The interaction of Hemin and Sestrin2 modulates oxidative stress and colon tumor growth.</a> Toxicology and Applied Pharmacology. 374, 77-85 (2019).</li><li>Wang, S. 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. 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=Troubleshooting+the+dichlorofluorescein+assay+to+avoid+artifacts+in+measurement+of+toxicant-stimulated+cellular+production+of+reactive+oxidant+species.">Troubleshooting the dichlorofluorescein assay to avoid artifacts in measurement of toxicant-stimulated cellular production of reactive oxidant species.</a> Journal of Pharmacological and Toxicological Methods. 67 (2), 56-60 (2013).</li><li>Rong, L., 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=Hydrogen+peroxide+detection+with+high+specificity+in+living+cells+and+inflamed+tissues.">Hydrogen peroxide detection with high specificity in living cells and inflamed tissues.</a> Regenerative Biomaterials. 3 (4), 217-222 (2016).</li><li>Liu, L. Z., 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=Quantitative+detection+of+hydroxyl+radical+generated+in+quartz+powder/phosphate+buffer+solution+system+by+fluorescence+spectrophotometry.">Quantitative detection of hydroxyl radical generated in quartz powder/phosphate buffer solution system by fluorescence spectrophotometry.</a> Guang Pu Xue Yu Guang Pu Fen Xi. 34 (7), 1886-1889 (2014).</li></ol>

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

JoVE Journal

Biochemistry

28.5K visualizações.  University of New Mexico. Este método pode ser usado para analisar espécies de oxigênio reativa celular em células aderentes com apenas um microscópio de fluorescência, e quantificar a intensidade das espécies de oxigênio reativa com leitor de placas de fluorescência. Este protocolo é simples, eficiente e econômico. Comece semeando duas vezes 10 para o quinto HCT116 células cancerígenas colorretais em DMEM em cada poço de uma placa de 24 poços.Incubar as células durante a noite a 37 graus Celsiu...