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 4.5 g/L D-glucose and 110 mg/L sodium pyruvate, add 10% heat-inactivated FBS, 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 2 mM L-glutamine, and 50 U/mL penicillin/streptomycin. Prepare the fresh medium, on the day the MIO-M1 cells will be thawed.</li>
	<li>Thaw the frozen MIO-M1 cells on day 1.
	<ol>
		<li>To do this, remove the cryovial containing 5 x 105 MIO-M1 cells in FBS with 10% DMSO from liquid nitrogen storage and immediately place it into a 37 &#176;C water bath. 
		NOTE: Always wear a face mask or safety goggles, since cryovials stored in liquid nitrogen present a risk of explosion when thawed.</li>
		<li>Thaw the MIO-M1 cells rapidly (in less than 1 min) by warming the vial in the 37 &#176;C water bath until there is just a small bit of ice left in the vial.</li>
		<li>Wipe the outside of the vial with 70% ethanol and transfer it to a laminar flow hood.</li>
		<li>Transfer the thawed cells from the vial to a sterile 15 mL tube and then add 6 mL of pre-warmed complete DMEM medium dropwise.</li>
		<li>Centrifuge the cell suspension at approximately 600 x g for 5 min. After the centrifugation, a clear supernatant and a complete pellet will be visualized. Discard the supernatant with a pipette without disturbing the cell pellet.</li>
		<li>Gently dissociate the pellet and resuspend the cells in 10 mL of complete DMEM medium. Transfer this cell suspension into a 100 mm diameter tissue culture plate and incubate the plate for approximately 2 days at 37 &#176;C, 5% CO2.</li>
	</ol>
	</li>
	<li>When the MIO-M1 cells become 80%-90% confluent on day 4, perform the following steps.
	<ol>
		<li>Transfer the plate into a laminar flow hood from the incubator. Carefully remove the supernatant and wash 2x with 5 mL of sterile and pre-warmed PBS. Aspirate the PBS.</li>
		<li>Dispense 1 mL of 0.5% Trypsin-EDTA solution and incubate at 37 &#176;C in the CO2&#160;incubator for 3-5 min to detach the cells.</li>
		<li>Add 7 mL of the complete DMEM medium to inhibit the trypsin action. Disaggregate clustered trypsinized MGCs by slowly pipetting up and down (P1000). Collect the cell suspension in a 15 mL tube.</li>
		<li>Centrifuge at 600 x g for 5 min. Discard the supernatant. Gently resuspend the cell pellet in 2 mL of complete DMEM media. Transfer 1 mL of this cell suspension into a 100 mm plate containing 9 mL of pre-warmed complete DMEM medium.</li>
		<li>Repeat the action with another 100 mm plate. Incubate the plate for approximately 1 day at 37 &#176;C in a 5% CO2&#160;incubator.</li>
	</ol>
	</li>
	<li>When both plates become 80%-90% confluent (on day 6), transfer the plate into a laminar flow hood. Follow step 1.3. to obtain a cell pellet.</li>
	<li>Gently dissociate the pellet and resuspend the cells in 5 mL of complete DMEM media. Count the MIO-M1 cells using a hemocytometer (Neubauer chamber) and trypan blue solution by following the steps below.
	<ol>
		<li>Put the glass cover on the Neubauer chamber central area. Place the chamber on a flat surface (e.g., a table or workbench).</li>
		<li>Mix an equal volume of trypan blue stain with the cells. For instance, mix 60 &#181;L of trypan blue with 60 &#181;L of cells for a 1:2 dilution.</li>
		<li>From this mixture, load 10 &#181;L with a micropipette into a Neubauer chamber.</li>
		<li>Place the loaded Neubauer chamber on the microscope stage. Then, turn on the light and adjust the brightness.</li>
		<li>Move the microscope stage to an optimal position and adjust the focus until a sharp image of the cells is obtained.</li>
		<li>Count the cells inside the four squares (subdivided in 16) located at the corner of the hemocytometer (volume: 0.1 mm3 each ).</li>
		<li>Calculate the cell concentration using the equations below. 
		Concentration = (Number of cells x 10.000)/Number of squares 
		Concentration = (Number of cells x 10.000)/4 
		Concentration = Number of cells x 2500 
		NOTE: If cell dilution is performed, the concentration obtained should be converted to the original concentration before the dilution. 
		&#8203;Concentration = (Number of cells x 2500 x 2) = (Number of cells x 5000 cells/mL)</li>
		<li>Adjust the cell suspension to 1 x 105 cells/mL in complete DMEM medium and plate 2 mL of this cell suspension into a 6-well plate (2 x 105&#160;cells/well). Shake the plate gently to distribute the cells homogeneously. Incubate the 6-well plate overnight at 37 &#176;C in a 5% CO2&#160;incubator.</li>
	</ol>
	</li>
</ol>
2. Assay conditions and controls
<ol>
	<li>Include the following experimental controls for each experiment:
	<ol>
		<li>Autofluorescence control (cells without DCFH-DA probe, control for setting the flow cytometer&#160;parameters).</li>
		<li>Basal control (unstimulated cells).</li>
		<li>Positive control (cells treated with a ROS inducer, A or B).</li>
		<li>Negative control (cells treated with antioxidant compound)</li>
		<li>Sample (cells treated with antioxidant compound and ROS inducer, A or B).</li>
	</ol>
	</li>
</ol>
3. Performing the assay
NOTE: The assay is performed on day 7. Always use proper aseptic technique and work in a laminar flow hood unless otherwise instructed.
<ol>
	<li>Prepare low serum DMEM. Supplement DMEM containing 4.5 g/L D-glucose and 110 mg/L sodium pyruvate with 0.5% heat-inactivated FBS,10 mM HEPES, 2 mM L-glutamine, and 50 U/mL penicillin/streptomycin. Prepare the fresh medium on the day the MIO-M1 will be treated.</li>
	<li>Transfer the 6-well plate (60%-70 % confluence) to a laminar flow hood from the incubator. Aspirate the supernatant and wash the cells 1x with PBS. Add 2 mL of the low serum DMEM per well and incubate the 6-well plate for 2 h at 37 &#176;C, 5% CO2. This is done to starve the cells.</li>
	<li>After serum starving, treat the cells with the antioxidant compound for 6 h.
	<ol>
		<li>Aspirate the supernatant and add 2 mL of the diluted stock to the following conditions: negative control (cells treated with antioxidant compound) and sample (cells treated with antioxidant compound and ROS inducer, A or B).</li>
		<li>Incubate the 6-well plate for 6 h at 37 &#176;C, 5% CO2. 
		NOTE: If using another antioxidant compound or ROS inhibitor, standardize the optimum time and concentration for the stimuli.</li>
	</ol>
	</li>
	<li>After 6 h of incubation, treat the cells with ROS inducer, A or B, for 30 min.
	<ol>
		<li>Add the stimuli to the following conditions: positive control (cells treated with a ROS inducer, A or B) and sample wells (cells treated with antioxidant compound and ROS inducer, A or B). 
		NOTE: Keep the stock solutions on ice.</li>
		<li>Incubate the 6-well plate for 30 min at 37 &#176;C, 5% CO2. 
		NOTE: If using another ROS inducer, properly standardize the optimum time and concentration for the stimuli.</li>
	</ol>
	</li>
	<li>Load the cells with the DCFH-DA probe.
	<ol>
		<li>Aspirate the supernatant and wash the cells in the 6-well plate 3x with PBS to remove all the stimuli.</li>
		<li>Turn off the light of the laminar flow hood and work in the dark. Prepare a dilution of 1:1000 of the 5 mM DCFH-DA probe stock solution to obtain a final concentration of 5 &#181;M DCFH-DA in DMEM without phenol red in a 15 mL tube.</li>
		<li>Mix gently using a vortex and add 1 mL of this solution to the following wells: basal control (unstimulated cells), positive control (cells treated with a ROS inducer), negative control (cells treated with antioxidant compound), and sample wells (cells treated with antioxidant compound, and ROS inducer, A or B).</li>
		<li>Add 1 mL of DMEM without phenol red to the autofluorescence control cells.</li>
		<li>Incubate the 6-well plate for 30 min at 37 &#176;C, 5% CO2. 
		&#8203;NOTE: The probe is light sensitive. Discard all the unused probe solution.</li>
	</ol>
	</li>
</ol>
4. Cell preparation for flow cytometry
<ol>
	<li>After 6 h, keep the plate on ice. From this step onward, it is not necessary to use proper aseptic technique and work in a laminar flow hood.</li>
	<li>Wash the cells 3x 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.</li>
	<li>Collect them in labeled 1.5 mL tubes and centrifuge at 600 x g for 5 min at 4 &#176;C. 
	NOTE: When harvesting the cells, avoid bubble formation by setting the P1000 pipette to 0.4 mL. From this step onward, work fast.</li>
	<li>When centrifugation ends, put labeled 1.5 mL tubes with the cells on ice. Discard the supernatant carefully and gently dissociate the cell pellet with tapping.</li>
	<li>Add 1 mL of FACS buffer to each labeled 1.5 mL tube to wash the cells. If necessary, use a vortex at its lowest intensity for the complete dissociation of the cell pellet. Centrifuge them at 600 x g for 5 min at 4 &#176;C. Repeat these steps 2x more. (Perform three washes in total).</li>
	<li>When the last wash ends, label 5 mL round-bottom&#160;polystyrene tubes (flow cytometry tubes) for all experimental conditions (see step 2.).</li>
	<li>When centrifugation ends, resuspend the cell pellets in 200 &#181;L of FACS buffer. Gently dissociate the pellet in each 1.5 mL tube by using a vortex. Transfer to labeled 5 mL round-bottom tubes. Keep the tubes on ice until they are analyzed on the flow cytometer.</li>
</ol>
5. Data acquisition in a flow cytometer
<ol>
	<li>Using the flow cytometry software, create a new experiment. To do this, in the Menu bar go to Experiment and click New Experiment (Ctrl+E).</li>
	<li>Also, in the Menu bar, go to View and click the following options: Toolbar, Status bar, Browser, Cytometer, Inspector, Worksheet, Acquisition Dashboard and Biexponential Editor to see these windows in the workspace.</li>
	<li>On the left of the software, click the Specimen_001 . This will open a list of tubes (Tube_001, Tube_002, etc.). To label the tubes for the controls and different experimental conditions, double-click Tube_001, Tube_002, etc. and rename them (see step 2.).</li>
	<li>Select the channels/parameters to be analyzed (FSC, SSC, FITC, and APC or any other fluorochrome to see the quadrant) in the first tube from Cytometer settings of the experiment.</li>
	<li>Click the Current Tube Pointer icon to select the tube, and the icon will change to green. Place the &#34;autofluorescence control&#34; tube on the aspirator arm, moving the base carefully only to the left.
	<ol>
		<li>To run the &#34;autofluorescence control&#34; sample, go to the Acquisition Dashboard window and click Acquire Data. Open a dot plot for FSC (on the x-axis) versus SSC (on the y-axis).</li>
		<li>Adjust forward and side scatter voltage quickly in order to ensure that the events appear on the graph. In the Acquisition Dashboard window click Stop Acquiring. In this experiment, the following voltage settings were used: FSC - 200 V, SSC - 423 V.</li>
	</ol>
	</li>
	<li>To set the FITC voltage, run the &#34;positive control (cells treated with a ROS inducer, A or B)&#34;. Adjust the voltage so that all the events appear on a histogram of FITC. In this case, use a voltage of 280 V. In the Acquisition Dashboard window, click Stop Acquiring.</li>
	<li>Place again the &#34;autofluorescence control&#34; tube on the aspirator arm. In the Acquisition Dashboard window, click Acquire Data, then Record Data, and select the desired stop conditions (for example, 50,000 events). Draw a gate around the cells of interest, excluding dead cells and debris. 
	NOTE: These are observed as much smaller events than the main cell population and appear on the lower left of the plot.</li>
	<li>When the acquisition ends, remove the tube from the aspirator arm. The equipment will wash itself. In the Acquisition Dashboard window, click Next Tube and run the Basal control (unstimulated cells). Click Acquire Data and then Record Data.
	<ol>
		<li>From the initial gate (gate that excludes the dead cells and debris), create another dot plot of FITC (on the x-axis) versus APC (on the y-axis). Draw a quadrant gate and adjust the coordinates in order to visualize the events on the lower left quadrant of the FITC versus APC plot.</li>
	</ol>
	</li>
	<li>For recording the next samples, remove the tube and click Next tube &#62; Acquire Data &#62; Record Data. 
	NOTE: The flow cytometer used here (see Table of Materials) records a maximum of 1 x 106 events per tube.</li>
	<li>When the recording of all tubes ends, export the data to disk D. To do this click File &#62; Export &#62; FCS files &#62; Select 3.0 or 3.1 version. 
	NOTE: Although data acquisition using one software (see Table of Materials) is described in detail in this protocol, any other flow cytometry software can be used. The acquisition parameters (FSC, SSC, and FITC) can be set in the same way to obtain the results.</li>
</ol>
6. Analysis of the acquired data
<ol>
	<li>Open the software and add the samples using the Add Samples action button; it is located on the taskbar or in the Navigate band.
	<ol>
		<li>Click Add Samples.</li>
		<li>Using the file browser, navigate to the Experimental folder.</li>
		<li>Select the Experimental folder and click Choose. 
		NOTE: The sample files load as part of the &#34;All Samples&#34; group into the workspace.</li>
	</ol>
	</li>
	<li>Double-click the first sample in the workspace: Autofluorescence Control. 
	NOTE: This action opens a Graph window plotting events along forward scatter (FSC-A on the x-axis) versus side scatter (SSC-A on the y-axis) parameters.</li>
	<li>Draw a polygon gate to isolate the cells of interest, excluding dead cells and debris.
	<ol>
		<li>Click the Polygon Gate tool.</li>
		<li>Click within the plot to make up the polygon gate. Make as many sides as needed.</li>
		<li>Double-click at the last point to close the polygon and create the gate desired.</li>
		<li>Provide a name for the gate, such as &#34;MIO-M1 cells&#34;, and press OK. 
		&#8203;NOTE: A new graph plot window appears displaying only the MIO-M1 events without dead cells and debris.</li>
		<li>Repeat with all the samples to analyze.</li>
	</ol>
	</li>
	<li>Change the plot to a histogram. To do so, click the X axis parameter label and select FITC-A. Click the Y axis parameter label and select Histogram. This changes the plot to a one-dimensional histogram.</li>
	<li>Add Statistics using the Add Statistic Window.
	<ol>
		<li>Below of histogram graph, click Add Statistics. The program will open a new statistic window.</li>
		<li>On the left of the new window select the statistics Geometric Mean.</li>
		<li>Select the Population MIO-M 1 cells.</li>
		<li>From the list of available parameters, select FIT-C.</li>
		<li>Click the Add button to apply them to the analysis. 
		NOTE: This creates the new statistics &#34;geometric mean: FITC&#34; in the sample gating tree, and their values are displayed in your workspace.</li>
	</ol>
	</li>
	<li>Put these geometric mean values in a statistics program and analyze.</li>
	<li>Click the L icon to open the Layout Editor; this icon is in Menu tab on the workspace. Position the Workspace window and Layout Editor side-by-side. 
	NOTE: The Layout Editor is a separated workspace window with tools for displaying multiple graph plots and statistics in a simple graphical report. This can be exported with the extension file you prefer, like PDF or JPG, among others.
	<ol>
		<li>From the gating tree of a sample in the Workspace window, drag MIO-M 1 cells population into the Layout Editor.</li>
		<li>Ensure that a histogram graph containing the events in the gate is displayed in the Layout Editor. Additional basic information will be detailed in a text box below.</li>
		<li>Drag all the samples to compare them in the same graph. The program will overlap them.</li>
		<li>Right-click the histogram graph and select Properties. The program will open a new Graph Definition window. This window has four tabs (Annotate, Fonts, Legend, and Specify). Click the Specify tab and change the y-axis from Auto to Modal. Click Apply.</li>
		<li>Right-click the sample and change Coloring, Line Style, Line Weight, etc. to personalize the graph.</li>
		<li>To export the image, click the File tab and, immediately after, select Save Image in the Document band. Choose the preferred type of image file (e.g., PDF). 
		NOTE: A save dialog box opens, prompting you to select a save location. Click Save.</li>
	</ol>
	</li>
	<li>Save the analysis as a workspace (.wsp) File.
	<ol>
		<li>On the top-left corner, click the Cytometry Analysis icon and select Save As.</li>
		<li>Choose Save as Workspace (WSP) to save the document with data and analysis.</li>
		<li>Enter a name for the workspace file.</li>
		<li>Click Save.</li>
	</ol>
	</li>
</ol>

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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. A., Ge, S. X., Studlack, P. E., Polster, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low+micromolar+concentrations+of+the+superoxide+probe+MitoSOX+uncouple+neural+mitochondria+and+inhibit+complex+IV.">Low micromolar concentrations of the superoxide probe MitoSOX uncouple neural mitochondria and inhibit complex IV.</a> Free Radical Biology and Medicine. 86, 250-258 (2015).</li></ol>

The authors have no conflicts of interest to disclose.

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 modulation of ROS are important targets for many diseases. The use of probes to measure ROS levels, like DCFH-DA, is accessible, widely described, and accepted in the scientific literature26,27,28. In this article, we describe a detailed and reproducible protocol to measure and quantify ROS levels by flow cytometry in MGCs.
An advantage of the use of this method is that, once the DCFH-DA probe is oxidized by ROS and generates a fluorescent DCF product; this can be measured in a plate reader, confocal microscope, or flow cytometer. The disadvantage of the plate reader is that it measures total fluorescence. Consequently, the plate reader does not distinguish the intracellular fluorescence from the extracellular one generated by chemical reactions in the culture medium. Confocal microscopy is a useful tool because cells can be loaded with DCFH-DA probe and viewed in real-time in culture chambers at 37&#176;C. The morphology and location of ROS in the cell can be detected with this methodology, but ROS levels lack a quantitative measurement. The strength of flow cytometry lies in its ability to measure the intracellular fluorescence in live cells. Quantitative data on the number of cells emitting fluorescence, as well as the geometric means of fluorescence, can be obtained25.
It is important to take into account what is actually measured when using DCFH-DA probe. As we previously mentioned, DCFH-DA measures multiple ROS species, and so fluorescence resulting from the green probe cannot be used to discriminate between types of ROS species29. In this regard, it is known that different probes can discern the type of ROS. For example, dihydroethidium (DHE) probe is oxidized by superoxide to yield the fluorescent product 2-hydroxyethidium (excitation at 518 nm and emission at 605 nm), but this product cannot be distinguished without the use of more time-consuming techniques, such as HPLC30,31. Another probe is a mitochondrial superoxide indicator (see Table of Materials), which is a novel fluorogenic probe for highly selective detection of superoxide in the mitochondria of living cells31,32.&#160;However, for the purposes of the measurement of total ROS levels and increases in ROS after an insult or the evaluation of the antioxidant ability of different products, the use of DCFH-DA probe alongside the appropriate controls is convenient and acceptable25,28,29.
A very important issue is the selection and use of proper experimental controls: autofluorescence control (cells without DCFH-DA probe), basal control (unstimulated cells), positive control (cells treated with a&#160;ROS inducer), negative control (cells treated with antioxidant compound), and sample problem (cells treated with antioxidant compound and ROS inducer). A useful tip is to use medium without phenol red to reduce interference with fluorescent probes.
We also recommend loading cells with the DCFH-DA probe 30 min before finishing the experimental treatment. Standardization of the concentration of DCFH-DA, in our case 5 &#181;M, is convenient and could differ depending on the cell type and the cell activation status. Our advice is to set up the probe concentrations, starting from 5&#181;M and 10 &#181;M, and check the efficacy of the staining in the experiments.
In summary, analyzing redox balance in health or disease is pivotal to establish reliable methods to measure the oxidative stress response. Therefore, this protocol is a simple, fast, and powerful tool to quantify ROS levels in live MGCs by flow cytometry.

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><tbody><tr><td>2&amp;prime;,7&amp;prime;-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&amp;rsquo;s modified Eagle&amp;rsquo;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>&amp;nbsp;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><td></td><td></td></tr></tbody></table>

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 the MIO-M1 cells. (Figure 1B, compare &#34;basal control&#34; vs. &#34;autofluorescence control&#34;, histogram graph).
Interestingly, a significant increase in FITC fluorescence was observed when the cells were stimulated with ROS inducer, A or B (Figure 1B, compare &#34;ROS inducer A&#34; samples vs. &#34;basal control&#34; and &#34;ROS inducer B&#34; samples vs. &#34;basal control&#34;, histogram graph). Moreover, when cells were treated with the antioxidant compound prior to ROS inducer, A or B, the fluorescence level was diminished near to basal levels (Figure 1B, compare &#34;ROS inducer A&#34; vs. &#34;pretreatment with antioxidant compound 6 h + ROS inducer A 30 min&#34;, and &#34;ROS inducer B&#34; vs. &#34;pretreatment with antioxidant compound 6 h + ROS inducer B 30 min&#34;, respectively, histogram graph). Of note, no differences in fluorescence signal were observed in cells treated with antioxidant compound (6 h) with respect to basal control. (Figure 1B, compare &#34;6h antioxidant compound&#34; vs. &#34;basal control&#34;).
These results were also clearly evident when data were presented as the geometric mean of FITC (Figure 1C). As can be seen in the graph, the average values and the corresponding standard error of the mean (SEM) are indicated.
A key advantage of this protocol is its application to follow changes in ROS over time. In this regard, we have been able to observe a significant and time-dependent increase in ROS when we treated MIO-M1 cells with ROS inducer C for 2 h, 4 h, and 6 h (Figure 2).
For optimization of this protocol, we have attempted three different strategies to harvest the cells (0.5% trypsin-EDTA, Accutase cell detachment solution, and detaching buffer) with comparable results (data not shown).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63337/63337fig01.jpg" /> 
Figure 1: Measurement of ROS in response to ROS inducer, A or B, using DCFH-DA probe in MIO-M1 cells: (A) Comparison of &#34;basal control&#34; vs. &#34;autofluorescence control&#34; conditions, representative dot plot FL1 (519 nm) vs. FL2 (660 nm). (B) Representative histograms for the fluorescence induced by DCFH-DA probe upon stimulation of MIO-M1 cells with ROS inducer, A or B, for 30 min, or pretreated with antioxidant compound for 6 h and 30 min of ROS inducer A, B, or vehicle. (C) Geometric mean of the FITC fluorescence for all the stimuli in MIO-M1 cells. Data are presented as mean &#177; SEM and analyzed by one-way ANOVA followed by Dunnett&#39;s post-test; **p &#60; 0.01, ***p &#60; 0.001. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63337/63337fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63337/63337fig02.jpg" /> 
Figure 2: Measurement of ROS overtime in response to ROS inducer C treatment using DCFH-DA probe in MIO-M1 cells: MIO-M1 cells treated with ROS inducer C for 2 h, 4 h, and 6 h and loaded with DCFH-DA probe to determine ROS levels. Representative histogram for fluorescence intensity induced by DCFH-DA probe upon stimulation of MIO-M1 cells with ROS inducer C for 2 h, 4 h, and 6 h. Geometric mean of FITC fluorescence for the stimuli detailed above. Data are presented as mean &#177; SEM and analyzed by one-way ANOVA followed by Dunnett&#39;s post-test; ns, not significant, **p &#60; 0.01, ***p &#60; 0.001. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63337/63337fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="2">Solution</td>
			<td></td>
			<td></td>
			<td>Storage conditions</td>
			<td colspan="3">Note</td>
		</tr>
		<tr>
			<td colspan="2">Sodium Hydroxide (NaOH) 10 M, 1 L</td>
			<td>400 g NaOH pellets</td>
			<td>distilled water to 1 L</td>
			<td>RT</td>
			<td colspan="3">&#8220;CAUTION&#8221;: The preparation of a concentrated NaOH solution cause an exothermic reaction. Extreme caution must be taken to avoid chemical burns and breakage of glass beakers. If possible, use heavy plastic beakers.</td>
		</tr>
		<tr>
			<td colspan="2">Phosphate-Buffered Saline (PBS) 10 X, 1 L</td>
			<td>80 g NaCl</td>
			<td>distilled water to 1 L</td>
			<td>RT</td>
			<td colspan="3">Adjust pH to 7.4 and filter the solution.</td>
		</tr>
		<tr>
			<td colspan="2"></td>
			<td>2 g KCl</td>
			<td></td>
			<td></td>
			<td colspan="3"></td>
		</tr>
		<tr>
			<td colspan="2"></td>
			<td>11.5 g Na2HPO4&#183; 7H2O</td>
			<td></td>
			<td></td>
			<td colspan="3"></td>
		</tr>
		<tr>
			<td colspan="2"></td>
			<td>2 g KH2PO4</td>
			<td></td>
			<td></td>
			<td colspan="3"></td>
		</tr>
		<tr>
			<td colspan="2">Ethylenediamine Tetraacetic Acid (EDTA), 0.5 M&#160;&#160;&#160; pH 8.0, 1 L</td>
			<td>186.1 g Na2EDTA&#183; 2H2O</td>
			<td>distilled water to 1 L</td>
			<td>RT</td>
			<td colspan="3">The disodium salt of EDTA is not soluble in neutral water or solution until the pH of the solution is adjusted to approximately 8.0 by the addition of NaOH. Adjust pH to 8.0 and sterilize the solution.</td>
		</tr>
		<tr>
			<td colspan="2">10% w/v Sodium Azide (NaN3), 100 mL</td>
			<td>10 g NaN3</td>
			<td>distilled water to 100 mL</td>
			<td>RT</td>
			<td colspan="3"></td>
		</tr>
		<tr>
			<td colspan="2">Detaching Buffer, 100 mL</td>
			<td>180 mg glucose</td>
			<td>PBS 1X to 100 mL</td>
			<td>4 &#176;C</td>
			<td colspan="3"></td>
		</tr>
		<tr>
			<td colspan="2"></td>
			<td>0.6 mL 0.5 M pH 8.0 EDTA</td>
			<td></td>
			<td></td>
			<td colspan="3"></td>
		</tr>
		<tr>
			<td colspan="2">Flow Cytometry Staining Buffer (FACS Buffer), 100 mL</td>
			<td>2 mL fetal bovine serum (FBS)</td>
			<td>PBS 1X to 100 mL</td>
			<td>4 &#176;C</td>
			<td colspan="3">NaN3 is added as a preservative.</td>
		</tr>
		<tr>
			<td colspan="2"></td>
			<td>1 mL 0.5 M pH 8.0 EDTA</td>
			<td></td>
			<td></td>
			<td colspan="3"></td>
		</tr>
		<tr>
			<td colspan="2"></td>
			<td>1 mL 10% w/v sodium azide</td>
			<td></td>
			<td></td>
			<td colspan="3"></td>
		</tr>
		<tr>
			<td colspan="2">5 mM 2&#8242;,7&#8242;-DCFH-DA, 1 mL</td>
			<td>2.4 mg DCFH-DA</td>
			<td>dimethyl sulfoxide to 1 mL</td>
			<td>-20 &#176;C protected from light</td>
			<td colspan="3">Mix gently and aliquot 20 &#956;L in 1.5 mL tubes. Avoid multiple thaw/freeze cycles. Our recommendation is discarding all unused probe solution from the experimental day.</td>
		</tr>
		<tr>
			<td colspan="2">0.4% w/v trypan blue 10 X, 50 mL</td>
			<td>0.2 g trypan blue</td>
			<td>PBS 1X to 50 mL</td>
			<td>4 &#176;C</td>
			<td colspan="3"></td>
		</tr>
	</tbody>
</table>
Table 1: Buffer recipes

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 ...

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6.5K Views.  Universidad Nacional de Córdoba. Er zijn verschillende methoden voor het meten van ROS-productie of oxidatieve stress in cellen. Onder deze, 2'7'dichlorofluoresceïne diacetaat sonde is een van de meest gebruikte techniek voor directe meting van redox toestand. Deze sonde is lipofiel en niet-fluorescerend. Diffusie van deze sonde over het celmembraan zorgt ervoor dat de intracellulaire esterasen zich splitsen bij de twee esterbindingen, waardoor een relatief polair en celmembraan-ondoordringbaar product ontstaat. Deze niet-f...

Video: Quantification of Reactive Oxygen Species Using 2′,7′-Dichlorofluorescein Diacetate Probe and Flow-Cytometry in Müller Glial Cells

Screening Assay for Oxidative Stress in a Feline Astrocyte Cell Line, G355-5

An often-suggested mechanism of virus induced neuronal damage is oxidative stress. Astrocytes have an important role in controlling oxidative stress of the Central Nervous System (CNS). Astrocytes help maintain a homeostatic environment for neurons as well as protecting neurons from Reactive Oxygen Species (ROS). CM-H2DCFDA is a cell-permeable indicator for the presence of ROS. CM-H2DCFDA enters the cell as a non-fluorescent compound, and becomes fluorescent after cellular esterases remove the acetate groups, and the compound is oxidized. The number of cells, measured by flow cytometry, that are found to be green fluorescing is an indication of the number of cells that are in an oxidative state. CM-H2DCFDA is susceptible to oxidation by a large number of different ROS. This lack of specificity, regarding which ROS can oxidize CM-H2DCFDA, makes this compound a valuable regent for use in the early stages of a pathogenesis investigation, as this assay can be used to screen for an oxidative cellular environment regardless of which oxygen radical or combination of ROS are responsible for the cellular conditions. Once it has been established that ROS are present by oxidation of CM-H2DCFDA, then additional experiments can be performed to determine which ROS or combination of ROSs are involved in the particular pathogenesis process. The results of this study demonstrate that with the addition of hydrogen peroxide an increase in CM-H2DCFDA fluoresce was detected relative to the saline controls, indicating that this assay is a valuable test for detecting an oxidative environment within G355-5 cells, a feline astrocyte cell line.

A screening method to detect oxidative cellular environments is to measure the oxidation of CM-H2DCFDA. Once oxidized within a cell, CM-H2DCFDA changes from non-fluorescent into a fluorescent compound. This change in fluorescence is measured by flow cytometry and indicates the number of cells in an oxidative environment.

An often-suggested mechanism of virus induced neuronal damage is oxidative stress. Astrocytes have an important role in controlling oxidative stress of ...

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.

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.

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

Biochemistry

Assessment of Cellular Oxidation using a Subcellular Compartment-Specific Redox-Sensitive Green Fluorescent Protein

Measuring the intracellular oxidation/reduction balance provides an overview of the physiological and/or pathophysiological redox status of an organism. Thiols are especially important for illuminating the redox status of cells via their reduced dithiol and oxidized disulfide ratios. Engineered cysteine-containing fluorescent proteins open a new era for redox-sensitive biosensors. One of them, redox-sensitive green fluorescent protein (roGFP), can easily be introduced into cells with adenoviral transduction, allowing the redox status of subcellular compartments to be evaluated without disrupting cellular processes. Reduced cysteines and oxidized cystines of roGFP have excitation maxima at 488 nm and 405 nm, respectively, with emission at 525 nm. Assessing the ratios of these reduced and oxidized forms allows the convenient calculation of redox balance within the cell. In this method article, immortalized human triple-negative breast cancer cells (MDA-MB-231) were used to assess redox status within the living cell. The protocol steps include MDA-MB-231 cell line transduction with adenovirus to express cytosolic roGFP, treatment with H2O2, and assessment of cysteine and cystine ratio with both flow cytometry and fluorescence microscopy.

This protocol describes the assessment of subcellular compartment-specific redox status within the cell. A redox-sensitive fluorescent probe allows convenient ratiometric analysis in intact cells.

Measuring the intracellular oxidation/reduction balance provides an overview of the physiological and/or pathophysiological redox status of an organism. ...

Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay

Microglia are the tissue resident macrophages of the central nervous system (CNS) and they perform a variety of functions that support CNS homeostasis, including phagocytosis of damaged synapses or cells, debris, and/or invading pathogens. Impaired phagocytic function has been implicated in the pathogenesis of diseases such as Alzheimer's and age-related macular degeneration, where amyloid-&#946; plaque and drusen accumulate, respectively. Despite its importance, microglial phagocytosis has been challenging to assess in vivo. Here, we describe a simple, yet robust, technique for precisely monitoring and quantifying the in vivo phagocytic potential of retinal microglia. Previous methods have relied on immunohistochemical staining and imaging techniques. Our method uses flow cytometry to measure microglial uptake of fluorescently labeled particles after intravitreal delivery to the eye in live rodents. This method replaces conventional practices that involve laborious tissue sectioning, immunostaining, and imaging, allowing for more precise quantification of microglia phagocytic function in just under six hours. This procedure can also be adapted to test how various compounds alter microglial phagocytosis in physiological settings. While this technique was developed in the eye, its use is not limited to vision research.

Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay

Microglial phagocytosis is critical for the maintenance of tissue homeostasis and inadequate phagocytic function has been implicated in pathology. However, assessing microglia function in vivo is technically challenging. We have developed a simple but robust technique for precisely monitoring and quantifying the phagocytic potential of microglia in a physiological setting.

Microglia are the tissue resident macrophages of the central nervous system (CNS) and they perform a variety of functions that support CNS homeostasis, ...

Immunology and Infection

Flow Cytometric Measurement Of ROS Production In Macrophages In Response To Fc&#947;R Cross-linking

The oxidative or respiratory burst is used to describe the rapid consumption of oxygen and generation of reactive oxygen species (ROS) by phagocytes in response to various immune stimuli. ROS generated during immune activation exerts potent antimicrobial activity primarily through the ability of ROS to damage DNA and proteins, causing death of microorganisms. Being able to measure ROS production reproducibly and with ease is necessary in order to assess the contribution of various pathways and molecules to this mechanism of host defense. In this paper, we demonstrate the use of fluorescent probes and flow cytometry to detect ROS production. Although widely used, fluorescent measurement of ROS is notoriously problematic, especially with regards to measurement of ROS induced by specific and not mitogenic stimuli. We present a detailed methodology to detect ROS generated as a result of specific Fc&#947;R stimulation beginning with macrophage generation, priming, staining, Fc&#947;R cross-linking, and ending with flow cytometric analysis.

This study demonstrates the use of flow cytometry to detect reactive oxygen species (ROS) production resulting from activation of the Fc&#947;R. This method can be used to assess changes in the antimicrobial and redox signaling function of phagocytes in response to immune complexes, opsonized microorganisms, or direct Fc&#947;R cross-linking.

The oxidative or respiratory burst is used to describe the rapid consumption of oxygen and generation of reactive oxygen species (ROS) by phagocytes in ...

Mouse Brain Histone Modification Quantification Using Flow Cytometry

Quantification of Global Histone Post Translational Modifications Using Intranuclear Flow Cytometry in Isolated Mouse Brain Microglia

Gene expression control occurs partially by modifications in chromatin structure, including the addition and removal of posttranslational modifications to histone tails. Histone post-translational modifications (HPTMs) can either facilitate gene expression or repression. For example, acetylation of histone tail lysine residues neutralizes the positive charge and reduces interactions between the tail and negatively charged DNA. The decrease in histone tail-DNA interactions results in increased accessibility of the underlying DNA, allowing for increased transcription factor access. The acetylation mark also serves as a recognition site for bromodomain-containing transcriptional activators, together resulting in enhanced gene expression. Histone marks can be dynamically regulated during cell differentiation and in response to different cellular environments and stimuli. While next-generation sequencing approaches have begun to characterize genomic locations for individual histone modifications, only one modification can be examined concurrently. Given that there are hundreds of different HPTMs, we have developed a high throughput, quantitative measure of global HPTMs that can be used to screen histone modifications prior to conducting more extensive genome sequencing approaches. This protocol describes a flow cytometry-based method to detect global HPTMs and can be conducted using cells in culture or isolated cells from in vivo tissues. We present example data from isolated mouse brain microglia to demonstrate the sensitivity of the assay to detect global shifts in HPTMs in response to a bacteria-derived immune stimulus (lipopolysaccharide). This protocol allows for the rapid and quantitative assessment of HPTMs and can be applied to any transcriptional or epigenetic regulator that can be detected by an antibody.

Author Spotlight: Enhancements in Gene Expression Regulation Research

This work describes a protocol for the quantification of global histone modifications using intranuclear flow cytometry in isolated brain microglia. The work also contains the microglia isolation protocol that was used for data collection.

Gene expression control occurs partially by modifications in chromatin structure, including the addition and removal of posttranslational modifications to ...

Flow Cytometry-Based Assays for Microglial Engulfment of Synaptic Materials

Quantification of Microglial Engulfment of Synaptic Material Using Flow Cytometry

Microglia play a pivotal role in synaptic refinement in the brain. Analysis of microglial engulfment of synapses is essential for comprehending this process; however, currently available methods for identifying microglial engulfment of synapses, such as immunohistochemistry (IHC) and imaging, are laborious and time-intensive. To address this challenge, herein we present in vitro and in vivo* assays that allow fast and high-throughput quantification of microglial engulfment of synapses using flow cytometry.
In the in vivo* approach, we performed intracellular vGLUT1 staining following fresh cell isolation from adult mouse brains to quantify engulfment of vGLUT1+ synapses by microglia. In the in vitro synaptosome engulfment assay, we used freshly isolated cells from the adult mouse brain to quantify the engulfment of pHrodo&#160;Red-labeled synaptosomes by microglia. These protocols together provide a time-efficient approach to quantifying microglial engulfment of synapses and represent promising alternatives to labor-intensive image analysis-based methods. By streamlining the analysis, these assays can contribute to a better understanding of the role of microglia in synaptic refinement in different disease models.

Author Spotlight: Advancing Understanding Through Technological Innovations in Psychoneuroimmunology

Here we present two protocols to quantify microglial engulfment of vGLUT1-positive synapses and pHRodo&#160;Red-labeled crude synaptosomes using flow cytometry.

Microglia play a pivotal role in synaptic refinement in the brain. Analysis of microglial engulfment of synapses is essential for comprehending this ...

Detecting Reactive Oxygen Species

Reactive oxygen species are chemically active, oxygen-derived molecules capable of oxidizing other molecules. Because of their reactive nature, there are many deleterious effects associated with unchecked ROS production, including structural damage to DNA and other biological molecules. However, ROS can also be mediators of physiological signaling. There is accumulating evidence that ROS play significant roles in everything from activation of transcription factors to the mediation of inflammatory toxicity that kills foreign pathogens and defend the body.In this video we will delve into the associations between ROS, metabolism and disease. After establishing their significance, we will discuss the principles and a protocol of a commonly used methodology for measuring ROS levels in cells: the use of non-fluorescent probes that become fluorescent upon oxidation. Lastly, we will review some current applications of this technique in cell biology research.

Reactive Oxygen Species and Oxidative Stress

Reactive oxygen species are chemically active, oxygen-derived molecules capable of oxidizing other molecules. Because of their reactive nature, there are ...

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Cell Biology

Fluorescent Probe Imaging Assay: A Technique to Visualize Oxidative Stress in the Reactive Oxygen Species Inducer-Treated Cultured Intestinal Organoid Cells

Source: Stedman, A. et al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/62880">Analyzing Oxidative Stress in Murine Intestinal Organoids using Reactive Oxygen Species-Sensitive Fluorogenic Probe.</a> J. Vis. Exp. (2021).
This video describes the technique of visualizing oxidative stress in cultured organoids. This work helps in understanding the level of oxidative stress due to ROS accumulation in the various cell types present in an organoid after treatment with different ROS modulators.

Source: Stedman, A. et al., Analyzing Oxidative Stress in Murine Intestinal Organoids using Reactive Oxygen Species-Sensitive Fluorogenic Probe. J. Vis. ...

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Cell-based assays

Flow Cytometry Assay to Quantify Oxidative Stress: An Assay to Quantify Reactive Oxygen Species in Intestinal Organoids Using Fluorogenic Probes

Source: Stedman, A., et al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/62880">Analyzing Oxidative Stress in Murine Intestinal Organoids using Reactive Oxygen Species-Sensitive Fluorogenic Probe.</a>&#160;J. Vis. Exp. (2021).
This video demonstrates a flow cytometry assay to quantify the ROS levels in 3D intestinal organoids obtained from GFP-expressing transgenic mice. This method provides an in vitro experimental model to investigate the efficacy of test compounds on ROS production.

Source: Stedman, A., et al., Analyzing Oxidative Stress in Murine Intestinal Organoids using Reactive Oxygen Species-Sensitive Fluorogenic Probe.&#160;J. ...

Quantification of Reactive Oxygen Species Using 2′,7′-Dichlorofluorescein Diacetate Probe and Flow-Cytometry in Müller Glial Cells

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