Name: quantification of reactive oxygen species using 2&#8242;,7&#8242;-dichlorofluorescein diacetate probe and flow-cytometry in m&#252;ller glial cells
Uploaded: 2022-05-13T14:00:00
Description: Watch this Scientific Journal Video about Quantification of Reactive Oxygen Species Using 2â€²,7â€²-Dichlorofluorescein Diacetate Probe and Flow-Cytometry in MÃ¼ller Glial Cells at JoVE.com

The authors would like to thank Mar&#237;a Pilar Crespo and Paula Alejandra Abadie of CIBICI (Centro de Investigaciones en Bioqu&#237;mica Cl&#237;nica e Inmunolog&#237;a, CONICET-UNC, C&#243;rdoba, Argentina) for assistance in flow cytometry and Gabriela Furlan and Noelia Maldonado for cell culture assistance. We also thank Victor Diaz (Pro-Secretary of Institutional Communication of FCQ) for the video production and editing.
This article was funded by grants from Secretar&#237;a de Ciencia y Tecnolog&#237;a, Universidad Nacional de C&#243;rdoba (SECyT-UNC) Consolidar 2018-2021, Fondo para la Investigaci&#243;n Cient&#237;fica y Tecnol&#243;gica (FONCyT), and Proyecto de Investigaci&#243;n en Ciencia y Tecnolog&#237;a (PICT) 2015 N&#176; 1314 (all to M.C.S.).

NOTE: For buffer compositions see Table 1.
1. Cell culture preparation
NOTE: Described here is the culture preparation of MIO-M1 cells, a spontaneously immortalized human M&#252;ller glial cell line (Moorfield&#39;s/Institute of Ophthalmology- M&#252;ller 1). Always use proper aseptic technique and work in a laminar flow hood.
<ol>
	<li>Prepare Dulbecco&#39;s modified Eagle&#39;s medium (DMEM) complete medium. To DMEM containing 4...

<ol>
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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signaling+pathways+triggered+by+oxidative+stress+that+mediate+features+of+severe+retinopathy+of+prematurity.">Signaling pathways triggered by oxidative stress that mediate features of severe retinopathy of prematurity.</a> JAMA Ophthalmology. 131 (1), 80-85 (2013).</li><li>Sies, H. <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:+a+concept+in+redox+biology+and+medicine.">Oxidative stress: a concept in redox biology and medicine.</a> Redox Biology. 4, 180-183 (2015).</li><li>Zorov, D. B., Juhaszova, M., Sollott, S. 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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=Nrf2+protects+photoreceptor+cells+from+photo-oxidative+stress+induced+by+blue+light.">Nrf2 protects photoreceptor cells from photo-oxidative stress induced by blue light.</a> Experiments in Eye Research. 154, 151-158 (2017).</li><li>Wei, Y., 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=Nrf2+in+ischemic+neurons+promotes+retinal+vascular+regeneration+through+regulation+of+semaphorin+6A.">Nrf2 in ischemic neurons promotes retinal vascular regeneration through regulation of semaphorin 6A.</a> Proceedings of the National Academy of Science of the United States of America. 112 (50), 6927-6936 (2015).</li><li>Wei, Y., 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=Nrf2+has+a+protective+role+against+neuronal+and+capillary+degeneration+in+retinal+ischemia-reperfusion+injury.">Nrf2 has a protective role against neuronal and capillary degeneration in retinal ischemia-reperfusion injury.</a> Free Radical Biology and Medicine. 51 (1), 216-224 (2011).</li><li>Wei, Y., 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=Nrf2+acts+cell-autonomously+in+endothelium+to+regulate+tip+cell+formation+and+vascular+branching.">Nrf2 acts cell-autonomously in endothelium to regulate tip cell formation and vascular branching.</a> Proceedings of the National Academy of Science of the United States of America. 110 (41), 3910-3918 (2013).</li><li>Wei, Y., Gong, J., Xu, Z., Duh, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nrf2+promotes+reparative+angiogenesis+through+regulation+of+NADPH+oxidase-2+in+oxygen-induced+retinopathy.">Nrf2 promotes reparative angiogenesis through regulation of NADPH oxidase-2 in oxygen-induced retinopathy.</a> Free Radical Biology and Medicine. 99, 234-243 (2016).</li><li>Xu, 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=Neuroprotective+role+of+Nrf2+for+retinal+ganglion+cells+in+ischemia-reperfusion.">Neuroprotective role of Nrf2 for retinal ganglion cells in ischemia-reperfusion.</a> Journal of Neurochemistry. 133 (2), 233-241 (2015).</li><li>Armstrong, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advanced+protocols+in+oxidative+stress+III.">Advanced protocols in oxidative stress III.</a> Methods in Molecular Biology. 1208, (2015).</li><li>Shehat, M. G., Tigno-Aranjuez, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometric+measurement+of+ROS+production+in+macrophages+in+response+to+Fc&#947;R+cross-linking.">Flow cytometric measurement of ROS production in macrophages in response to Fc&#947;R cross-linking.</a> Journal of Visualized Experiments. (145), e59167 (2019).</li><li>Wu, D., Yotnda, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+and+detection+of+reactive+oxygen+species+(ROS)+in+cancers.">Production and detection of reactive oxygen species (ROS) in cancers.</a> Journal of Visualized Experiments. (57), e3357 (2011).</li><li>Halliwell, B., Whiteman, M. <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+species+and+oxidative+damage+in+vivo+and+in+cell+culture:+how+should+you+do+it+and+what+do+the+results+mean.">Measuring reactive species and oxidative damage in vivo and in cell culture: how should you do it and what do the results mean.</a> British Journal of Pharmacology. 142 (2), 231-255 (2004).</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 Radical Biology and Medicine. 52 (1), 1-6 (2012).</li><li>Fernandes, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+DHE-derived+oxidation+products+by+HPLC+in+the+assessment+of+superoxide+production+and+NADPH+oxidase+activity+in+vascular+systems.">Analysis of DHE-derived oxidation products by HPLC in the assessment of superoxide production and NADPH oxidase activity in vascular systems.</a> American Journal of Physiology and Cell Physiology. 292 (1), 413-422 (2007).</li><li>Zielonka, J., Kalyanaraman, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydroethidine-+and+MitoSOX-derived+red+fluorescence+is+not+a+reliable+indicator+of+intracellular+superoxide+formation:+another+inconvenient+truth.">Hydroethidine- and MitoSOX-derived red fluorescence is not a reliable indicator of intracellular superoxide formation: another inconvenient truth.</a> Free Radical Biology and Medicine. 48 (8), 983-1001 (2010).</li><li>Roelofs, B. 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Several pathological conditions, such as cancer, inflammatory diseases, ischemia/reperfusion, ischemic heart disease, diabetes, and retinopathies, and also physiological situations like aging, lead to ROS overproduction6,7,8,9,10,11. Therefore, the detection, measurement, and understanding of the pathway involved in the modul...

The neural retina is a very organized tissue that presents well-defined neuronal layers. In these, neurons (ganglion, amacrine, bipolar, horizontal, and photoreceptor cells) are interconnected to each other and also with M&#252;ller glial cells (MGCs) and astrocytes, leading to adequate phototransduction and processing of visual information1,2. MGCs are known to have an important role in the maintenance of retinal homeostasis because they cross the entire retinal section and, thus, they can interact with all cell types that modulate multiple protective processes. It has been reported that MGCs have several important functions for the maintenance and survival of retinal neurons, including glycolysis to provide energy to neurons, the removal of neuronal waste, the recycling of neurotransmitters, and the release of neurotrophic factors, among others3,4,5.
On the other hand, inflammation, oxidative and nitrosative stress are involved in the pathogenesis and progression of many human diseases, including retinopathies6,7,8,9,10,11. The redox balance in cells depends on tight regulation of ROS levels. ROS are constantly generated under physiological conditions as a result of aerobic respiration mainly. The major members of the ROS family include reactive free radicals such as the superoxide anion (O2&#856;&#856;&#856;&#856;&#8226;&#8722;), hydroxyl radicals (&#8226;OH), various peroxides (ROOR&#8242;), hydroperoxides (ROOH), and the no radical hydrogen peroxide (H2O2)12,13. In the last years, it has become apparent that ROS plays an important signaling role in the cells by controlling essential processes. MGCs have a strong antioxidant defense by the activation of the transcriptional nuclear factor erythroid-2-related factor 2 (Nrf2) and the subsequent expression of antioxidant proteins to eliminate the excessive production of ROS under pathological conditions14,15,16. When the cells lose their redox balance due to an exaggerated production of ROS or a defective ability to remove ROS, the accumulation of oxidative stress promotes harmful modifications in proteins, lipids, and DNA, leading to cellular stress or death. The increase of the retinal antioxidant defense system improves the resolution and prevention of retinopathies, such as ROP and RD17,18,19,20,21,22,23,24. Therefore, the measurement of ROS production in real-time is a powerful and useful tool.
There are several methods for measuring ROS production or oxidative stress in cells. Among these, 2&#8242;,7&#8242;-dichlorofluorescein diacetate (DCFH-DA) probe is one of the most widely used techniques for directly quantifying the redox state of a cell25,26,27,28. This probe is lipophilic and non-fluorescent. Diffusion of this probe across the cell membrane allows its cleavage by intracellular esterases at the two ester bonds, producing a relatively polar and cell membrane-impermeable product, 2&#8242;,7&#8242;-dichlorofluorescein (H2DCF). This non-fluorescent molecule accumulates intracellularly, and subsequent oxidation by ROS yields the highly fluorescent product DCF. The oxidation of the probe is the product of the action of multiple types of ROS (peroxynitrite, hydroxyl radicals, nitric oxide, or peroxides), which can be detected by flow cytometry or confocal microscopy (emission at 530 nm and excitation at 485 nm). The limitation of this technique is that superoxide and hydrogen peroxide do not strongly react with H2DCF25,29. In this article, we use DCFH-DA probe to measure and quantify ROS by flow cytometry. For that reason, we induce ROS production by stimulating MGCs with ROS inducer, A or B, previous to loading the cells with the fluorescent probe. In addition, we use an antioxidant compound. Finally, we show representative and reliable data obtained using this protocol.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>2&#8242;,7&#8242;-DCFH-DA</td>
			<td>Sigma</td>
			<td>35845-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)</td>
			<td>Gibco by life technologies</td>
			<td>15630-080</td>
			<td></td>
		</tr>
		<tr>
			<td>BD FACSCanto II flow cytometer</td>
			<td>BD Biosciences</td>
			<td>FACSCanto</td>
			<td></td>
		</tr>
		<tr>
			<td>BD FACSDiva software</td>
			<td>BD Biosciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Culture Dishes 100x20 mm</td>
			<td>Cell Star- Greiner Bio-One</td>
			<td>664 160</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Thermo</td>
			<td>Sorvall legend micro 17 R</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge Tubes (15 ml)</td>
			<td>BIOFIL</td>
			<td>CFT011150</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge Tubes (50 ml)</td>
			<td>BIOFIL</td>
			<td>CFT011500</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryovial</td>
			<td>CRYO.S - Greiner Bio-One</td>
			<td>126263</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl Sulfoxide</td>
			<td>Sigma-Aldrich</td>
			<td>W387520-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Disodium-hydrogen-phosphate heptahydrate</td>
			<td>Merck</td>
			<td>106575</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM without phenol red</td>
			<td>Gibco by life technologies</td>
			<td>31053-028</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s modified Eagle&#8217;s medium (DMEM)</td>
			<td>Gibco by life technologies</td>
			<td>11995065</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylenediamine Tetraacetic Acid (EDTA), Disodium Salt, Dihydrate</td>
			<td>Merck</td>
			<td>324503</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Internegocios</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>FlowJo v10 Software</td>
			<td>BD Biosciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Merck</td>
			<td>108337</td>
			<td></td>
		</tr>
		<tr>
			<td>hemocytometer, Neubauer chamber</td>
			<td>BOECO,Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Laminar flow hood</td>
			<td>ESCO</td>
			<td>AC2-6E8</td>
			<td></td>
		</tr>
		<tr>
			<td>L-glutamine (GlutaMAX)</td>
			<td>Gibco by life technologies</td>
			<td>A12860-01</td>
			<td></td>
		</tr>
		<tr>
			<td>MitoSOX Red</td>
			<td>Invitrogen</td>
			<td>&#160;M36008</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Gibco by life technologies</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Chloride</td>
			<td>Merck</td>
			<td>104936</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium-dihydrogen phosphate</td>
			<td>Merck</td>
			<td>4878</td>
			<td></td>
		</tr>
		<tr>
			<td>Round polystyrene tubes 5 ml (flow cytometry tubes)</td>
			<td>Falcon - Corning</td>
			<td>BD-352008</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Azide</td>
			<td>Merck</td>
			<td>822335</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Merck</td>
			<td>106404</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Hydroxide</td>
			<td>Merck</td>
			<td>106462</td>
			<td></td>
		</tr>
		<tr>
			<td>SPINWIN Micro Centrifuge Tube 1.5 ml</td>
			<td>Tarson</td>
			<td>500010-N</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue Culture Plate 6 well</td>
			<td>BIOFIL</td>
			<td>TCP011006</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue</td>
			<td>Merck</td>
			<td>111732</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA 0.5% 10X</td>
			<td>Gibco by life technologies</td>
			<td>15400-054</td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex Mixer</td>
			<td>Labnet International, Inc.</td>
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quantification of reactive oxygen species using 2&#8242;,7&#8242;-dichlorofluorescein diacetate probe and flow-cytometry in m&#252;ller glial cells

The redox balance has an important role in maintaining cellular homeostasis. The increased generation of reactive oxygen species (ROS) promotes the modification of proteins, lipids, and DNA, which finally may lead to alteration in cellular function and cell death. Therefore, it is beneficial for cells to increase their antioxidant defense in response to detrimental insults, either by activating an antioxidant pathway like Keap1/Nrf2 or by improving redox scavengers (vitamins A, C, and E, &#946;-carotene, and polyphenols, among others). Inflammation and oxidative stress are involved in the pathogenesis and progression of retinopathies, such as diabetic retinopathy (DR) and retinopathy of prematurity (ROP). Since M&#252;ller glial cells (MGCs) play a key role in the homeostasis of neural retinal tissue, they are considered an ideal model to study these cellular protective mechanisms. In this sense, quantifying ROS levels with a reproducible and simple method is essential to assess the contribution of pathways or molecules that participate in the antioxidant cell defense mechanism. In this article, we provide a complete description of the procedures required for the measurement of ROS with DCFH-DA probe and flow cytometry in MGCs. Key steps for flow cytometry data processing with the software are provided here, so the readers will be able to measure ROS levels (geometric means of FITC) and analyze fluorescence histograms. These tools are highly helpful to evaluate not only the increase in ROS after a cellular insult but also to study the antioxidant effect of certain molecules that can provide a protective effect on the cells.

As described in the protocol section, we have shown representative and quantitative data demonstrating flow cytometry detection of ROS production with the fluorescence probe DCFH-DA from MIO-M1 cells stimulated with ROS inducer, A or B. As expected, we observed changes in FITC fluorescence in unstimulated cells above autofluorescence levels (Figure 1A, compare &#34;basal control&#34; vs. &#34;autofluorescence control&#34;, dot plots graph). This occurred due to a basal production of ROS in t...

Watch this Scientific Journal Video about Quantification of Reactive Oxygen Species Using 2â€²,7â€²-Dichlorofluorescein Diacetate Probe and Flow-Cytometry in MÃ¼ller Glial Cells at JoVE.com

Quantification of Reactive Oxygen Species Using 2&#8242;,7&#8242;-Dichlorofluorescein Diacetate Probe and Flow-Cytometry in M&#252;ller Glial Cells

Here, we propose a systematized, accessible, and reproducible protocol to detect cellular reactive oxygen species (ROS) using 2&#8242;,7&#8242;-dichlorofluorescein diacetate probe (DCFH-DA) in M&#252;ller glial cells (MGCs). This method quantifies total cellular ROS levels with a flow cytometer. This protocol is very easy to use, suitable, and reproducible.

The redox balance has an important role in maintaining cellular homeostasis. The increased generation of reactive oxygen species (ROS) promotes the ...

There are several methods for measure ROS production, or oxidative stress in cells.Among these, 2'7'dichlorofluorescein diacetate probe is one of the most widely used technique for directly measure of redox state.This probe is lipophilic and non-fluorescent.Diffusion of this probe across the cell membrane allow the intracellular esterases cleavage at the two ester bonds, producing a relatively polar and cell membrane-impermeable product.These non-fluorescent molecules accumulate intracellularly, and subsequent oxidation by ROS yields the highly fluorescent product dichlorofluorescein.The oxidation of the probe is the product of action of multiple type of ROS.Which can be detected by flow cytometry or confocal microscopy.In this article, we have used a dichlorofluorescein diacetate probe to measure and quantify ROS by flow cytometry.Transfer 6-well plates into a laminar flow hood from the incubator.Aspirate the supernatant.And wash the cells once with PBS.Add 2 mL of low serum DMEM per well.And incubate the 6-well plate for two hours in the incubator.Treat the cells with the antioxidant for six hours.Treat the cells with the ROS inducer, A or B, for 30 minutes.Aspirate the supernatant.And wash the cell three times with PBS to remove all the stimuli.Turn off the light of the laminar flow hood and work in darkness.Add 1 mL of dichlorofluorescein diacetate probe in DMEM without phenol red.Add 1 mL of DMEM without phenol red to the control autofluorescence control cells.Incubate the 6-well plate for 30 minutes in the incubator.Transfer the plates on ice to the laboratory.Wash the cells three times with 2 mL of cold PBS per well.Add 0.5 mL of cold detaching buffer to each well.Harvest the cells by gently pipetting up and down using a P1000 pipette.Collect them in labeled 1.5 mL tube.And centrifuge them at 600 x g for five minutes at 4 C.Discard the supernatant carefully, and gently dissociate the pellet with tapping.Add 1 mL of FACS buffer to wash the cells to each labeled tube.If it's necessary, use vortex in the lowest intensity for the complete dissociation of the pellet.Centrifuge them at 600 x g for five minutes at 4 C.Repeat this step two times more.Three washes in total.Resuspend cell pellets in 200 L of FACS buffer.Gently dissociate completely the pellet in each tube by using vortex.And transfer to labeled 5 mL for flow cytometer tubes.Keep the tubes on ice until analyze on flow cytometer.Using the flow cytometry software, create a new experiment.Click the specimen and will open a list of tubes.Double-click and rename them.Place the autofluorescence control tube on the aspirator arm, moving the base carefully only to the left.Click Acquire Data and then Record Data, and select the desired stop condition.Draw a gate around the cells of interest, excluding dead cells and debris.Remove the tube.Click Next Tube and run the basal control sample.Click Acquire Data and then in Record Data.Open the software.Add the samples using the Add Samples action button.Click Add Sample.Select the Experimental Date folder and click Choose.Double-click the first sample in your workspace, autofluorescence control in this case.Draw a polygon gate to isolate the cell of interest, excluding dead cells and debris.Change plot to a histogram.Double-click within the M

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