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 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>
			<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 t...

Watch this Scientific Journal Video about Quantification of Reactive Oxygen Species Using 2Ã¢â‚¬Â²,7Ã¢â‚¬Â²-Dichlorofluorescein Diacetate Probe and Flow-Cytometry in MÃƒÂ¼ller 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 ...

quantification-reactive-oxygen-species-using-27-dichlorofluorescein

Research

JoVE Journal

Neuroscience

6.3K Views.  Universidad Nacional de Córdoba. Here, we propose a systematized, accessible, and reproducible protocol to detect cellular reactive oxygen species (ROS) using 2′,7′-dichlorofluorescein diacetate probe (DCFH-DA) in Müller glial cells (MGCs). This method quantifies total cellular ROS levels with a flow cytometer. This protocol is very easy to use, suitable, and reproducible. 

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

Determination of Mitochondrial Membrane Potential and Reactive Oxygen Species in Live Rat Cortical Neurons

Mitochondrial membrane potential (&Delta;&Psi;m) is critical for maintaining the physiological function of the respiratory chain to generate ATP. A significant loss of &Delta;&Psi;m renders cells depleted of energy with subsequent death. Reactive oxygen species (ROS) are important signaling molecules, but their accumulation in pathological conditions leads to oxidative stress. The two major sources of ROS in cells are environmental toxins and the process of oxidative phosphorylation. Mitochondrial dysfunction and oxidative stress have been implicated in the pathophysiology of many diseases; therefore, the ability to determine &Delta;&Psi;m and ROS can provide important clues about the physiological status of the cell and the function of the mitochondria. 
Several fluorescent probes (Rhodamine 123, TMRM, TMRE, JC-1) can be used to determine &Delta;&psi;m in a variety of cell types, and many fluorescence indicators (Dihydroethidium, Dihydrorhodamine 123, H2DCF-DA) can be used to determine ROS. Nearly all of the available fluorescence probes used to assess &Delta;&Psi;m or ROS are single-wavelength indicators, which increase or decrease their fluorescence intensity proportional to a stimulus that increases or decreases the levels of &Delta;&Psi;m or ROS. Thus, it is imperative to measure the fluorescence intensity of these probes at the baseline level and after the application of a specific stimulus. This allows one to determine the percentage of change in fluorescence intensity between the baseline level and a stimulus. This change in fluorescence intensity reflects the change in relative levels of &Delta;&Psi;m or ROS. In this video, we demonstrate how to apply the fluorescence indicator, TMRM, in rat cortical neurons to determine the percentage change in TMRM fluorescence intensity between the baseline level and after applying FCCP, a mitochondrial uncoupler. The lower levels of TMRM fluorescence resulting from FCCP treatment reflect the depolarization of mitochondrial membrane potential. We also show how to apply the fluorescence probe H2DCF-DA to assess the level of ROS in cortical neurons, first at baseline and then after application of H2O2. This protocol (with minor modifications) can be also used to determine changes in &#x2206;&#x03A8;m and ROS in different cell types and in neurons isolated from other brain regions.

We demonstrate application of the fluorescence indicator, TMRM, in cortical neurons to determine the relative changes in TMRM fluorescence intensity before and after application of a specific stimulus. We also show application of the fluorescence probe H2DCF-DA to assess the relative level of reactive oxygen species in cortical neurons.

Mitochondrial membrane potential (&Delta;&Psi;m) is critical for maintaining the physiological function of the respiratory chain to generate ATP.  A ...

Isolating LacZ-expressing Cells from Mouse Inner Ear Tissues using Flow Cytometry

Isolation of specific cell types allows one to analyze rare cell populations such as stem/progenitor cells. Such an approach to studying inner ear tissues presents a unique challenge because of the paucity of cells of interest and few transgenic reporter mouse models. Here, we describe a protocol using fluorescence-conjugated probes to selectively label LacZ-positive cells from the neonatal cochleae.
The most common underlying pathology of sensorineural hearing loss is the irreversible damage and loss of cochlear sensory hair cells, which are required to transduce sound waves to neural impulses. Recent evidence suggests that the murine auditory and vestibular organs harbor stem/progenitor cells that may have regenerative potential1,2. These findings warrant further investigation, including identifying specific cell types with stem/progenitor cell characteristics. The Wnt signaling pathway has been demonstrated to play a critical role in maintaining stem/progenitor cell populations in several organ systems3-7. We have recently identified Wnt-responsive Axin2-expressing cells in the neonatal cochlea, but their function is largely unknown8.
To better understand the behavior of these Wnt-responsive cells in vitro, we have developed a method of isolating Axin2-expressing cells from cochleae of Axin2-LacZ reporter mice9. Using flow cytometry to isolate Axin2-LacZ positive cells from the neonatal cochleae, we could in turn execute a variety of experiments on live cells to interrogate their behavior as stem/progenitor cells. Here, we describe in detail the steps for the microdissection of neonatal cochlea, dissociation of these tissues, labeling of the LacZ-positive cells using a fluorogenic substrate, and cell sorting. Techniques for dissociating cochleae into single cells and isolating cochlear cells via flow cytometry have been described2,10-12. We have made modifications to these techniques to establish a novel protocol to isolate LacZ-expressing cells from the neonatal cochlea.

Flow cytometry is a powerful tool allowing for the isolation and study of specific cell populations. This protocol describes steps for isolating LacZ-expressing cells from cochlear tissues from neonatal transgenic mice. Dissociated cochlear cells were labeled using fluorescent-conjugated substrates of &#946;-galactosidase prior to separation via flow cytometry.

Isolation of specific cell types allows one to analyze rare cell populations such as stem/progenitor cells. Such an approach to studying inner ear tissues ...

The Neuroblast Assay: An Assay for the Generation and Enrichment of Neuronal Progenitor Cells from Differentiating Neural Stem Cell Progeny Using Flow Cytometry

Neural stem cells (NSCs) can be isolated and expanded in large-scale, using the neurosphere assay and differentiated into the three major cell types of the central nervous system (CNS); namely, astrocytes, oligodendrocytes and neurons. These characteristics make neural stem and progenitor cells an invaluable renewable source of cells for in vitro studies such as drug screening, neurotoxicology and electrophysiology and also for cell replacement therapy in many neurological diseases. In practice, however, heterogeneity of NSC progeny, low production of neurons and oligodendrocytes, and predominance of astrocytes following differentiation limit their clinical applications. Here, we describe a novel methodology for the generation and subsequent purification of immature neurons from murine NSC progeny using fluorescence activated cell sorting (FACS) technology. Using this methodology, a highly enriched neuronal progenitor cell population can be achieved without any noticeable astrocyte and bona fide NSC contamination. The procedure includes differentiation of NSC progeny isolated and expanded from E14 mouse ganglionic eminences using the neurosphere assay, followed by isolation and enrichment of immature neuronal cells based on their physical (size and internal complexity) and fluorescent properties using flow cytometry technology. Overall, it takes 5-7 days to generate neurospheres and 6-8 days to differentiate NSC progeny and isolate highly purified immature neuronal cells.

This video protocol demonstrates a novel method for the generation and subsequent purification of neuronal progenitor cells from a renewable source of neural stem cells (NSCs) based on their physical (size and internal granularity) and fluorescent properties using flow cytometry technology.

Neural stem cells (NSCs) can be isolated and expanded in large-scale, using the neurosphere assay and differentiated into the three major cell types of ...

Inducing Plasticity of Astrocytic Receptors by Manipulation of Neuronal Firing Rates

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be stimulated by neuronally-released transmitter. However, the ability of astrocytic receptors to exhibit plasticity in response to changes in neuronal activity has received little attention. Here we describe a model system that can be used to globally scale up or down astrocytic group I metabotropic glutamate receptors (mGluRs) in acute brain slices. Included are methods on how to prepare parasagittal hippocampal slices, construct chambers suitable for long-term slice incubation, bidirectionally manipulate neuronal action potential frequency, load astrocytes and astrocyte processes with fluorescent Ca2+ indicator, and measure changes in astrocytic Gq GPCR activity by recording spontaneous and evoked astrocyte Ca2+ events using confocal microscopy. In essence, a &#8220;calcium roadmap&#8221; is provided for how to measure plasticity of astrocytic Gq GPCRs. Applications of the technique for study of astrocytes are discussed. Having an understanding of how astrocytic receptor signaling is affected by changes in neuronal activity has important implications for both normal synaptic function as well as processes underlying neurological disorders and neurodegenerative disease.

Here we describe an adaptation of protocols used to induce homeostatic plasticity in neurons for the study of plasticity of astrocytic G protein-coupled receptors. Recently used to examine changes in astrocytic group I mGluRs in juvenile mice, the method can be applied to measure scaling of various astrocytic GPCRs, in tissue from adult mice in situ and in vivo, and to gain a better appreciation of the sensitivity of astrocytic receptors to changes in neuronal activity.

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be ...

Assessment of Neuronal Viability Using Fluorescein Diacetate-Propidium Iodide Double Staining in Cerebellar Granule Neuron Culture

Primary cultured Cerebellar Granule Neurons (CGNs) have been widely used as an in vitro model in neuroscience and neuropharmacology research. However, the co-existence of glial cells and neurons in CGN culture might lead to biases in the accurate assessment of neuronal viability. Fluorescein diacetate (FDA) and Propidium Iodide (PI) double staining has been used to measure cell viability by simultaneously evaluating the viable and dead cells. We used FDA-PI double staining to improve the sensitivities of the colorimetric assays and to evaluate neuronal viability in CGNs. Furthermore, we added blue fluorescent DNA stains (e.g., Hoechst) to improve the accuracy. This protocol describes how to improve the accuracy of assessment of neuronal viability by using these methods in CGN culture. Using this protocol, the number of glial cells can be excluded by using fluorescence microscopy. A similar strategy can be applied to distinguish the unwanted glial cells from neurons in various mixed cell cultures, such as primary cortical culture and hippocampal culture.

This protocol describes how to accurately measure neuronal viability using Fluorescein diacetate (FDA) and Propidium Iodide (PI) double staining in cultured cerebellar granule neurons, a primary neuronal culture used as an in vitro model in neuroscience and neuropharmacology research.

Primary cultured Cerebellar Granule Neurons (CGNs) have been widely used as an in vitro model in neuroscience and neuropharmacology research. However, the ...

Quantification of Endosome and Lysosome Motilities in Cultured Neurons Using Fluorescent Probes

In the brain, membrane trafficking systems play important roles in regulating neuronal functions, such as neuronal morphology, synaptic plasticity, survival, and glial communications. To date, numerous studies have reported that defects in these systems cause various neuronal diseases. Thus, understanding the mechanisms underlying vesicle dynamics may provide influential clues that could aid in the treatment of several neuronal disorders. Here, we describe a method for quantifying vesicle motilities, such as motility distance and rate of movement, using a software plug-in for the ImageJ platform. To obtain images for quantification, we labeled neuronal endosome-lysosome structures with EGFP-tagged vesicle marker proteins and observed the movement of vesicles using a time-lapse microscopy. This method is highly useful and simplify measuring vesicle motility in neurites, such as axons and dendrites, as well as in the soma of both neurons and glial cells. Furthermore, this method can be applied to other cell lines, such as fibroblasts and endothelial cells. This approach could provide a valuable advancement of our understanding of membrane trafficking.

The investigation of membrane trafficking is crucial for understanding neuronal functions. Here, we introduce a method for quantifying vesicle motility in neurons. This is a convenient method that can be adapted to the quantification of membrane trafficking in the nervous system.

In the brain, membrane trafficking systems play important roles in regulating neuronal functions, such as neuronal morphology, synaptic plasticity, ...

Analysis of Immune Cells in Single Sciatic Nerves and Dorsal Root Ganglion from a Single Mouse Using Flow Cytometry

Nerve-resident immune cells in the peripheral nervous system (PNS) are essential to maintaining neuronal integrity in a healthy nerve. The immune cells of the PNS are affected by injury and disease, affecting the nerve function and the capacity for regeneration. Neuronal immune cells are commonly analyzed by immunofluorescence (IF). While IF is essential for determining the location of the immune cells in the nerve, IF is only semi-quantitative and the method is limited to the number of markers that can be analyzed simultaneously and the degree of surface expression. In this study, flow cytometry was used for quantitative analysis of leukocyte infiltration into sciatic nerves or dorsal root ganglions (DRGs) of individual mice. Single cell analysis was performed using DAPI and several proteins were analyzed simultaneously for either surface or intracellular expression. Both sciatic nerves from one mouse that were treated according to this protocol generated &#8805; 30,000 single nucleated events. The proportion of leukocytes in the sciatic nerves, determined by expression of CD45, was approximately 5% of total cell content in the sciatic nerve and approximately 5-10% in the DRG. Although this protocol focuses primarily on the immune cell population within the PNS, the flexibility of flow cytometry to measure a number of markers simultaneously means that the other cells populations present within the nerve, such as Schwann cells, pericytes, fibroblasts, and endothelial cells, can also be analyzed using this method. This method therefore provides a new means for studying systemic effects on the PNS, such as neurotoxicology and genetic models of neuropathy or in chronic diseases, such as diabetes.

Quantitative analysis of cell content within the murine sciatic nerve is difficult due to the scarcity of the tissue. This protocol describes a method for tissue digestion and preparation that provides sufficient cells for flow cytometry analysis of immune cell populations from nerves of individual mice.

Nerve-resident immune cells in the peripheral nervous system (PNS) are essential to maintaining neuronal integrity in a healthy nerve. The immune cells of ...

Using Optical Coherence Tomography and Optokinetic Response As Structural and Functional Visual System Readouts in Mice and Rats

Optical coherence tomography (OCT) is a fast, non-invasive, interferometric technique allowing high-resolution retinal imaging. It is an ideal tool for the investigation of processes of neurodegeneration, neuroprotection and neuro-repair involving the visual system, as these often correlate well with retinal changes. As a functional readout, visually evoked compensatory eye and head movements are commonly used in experimental models involving the visual function. Combining both techniques allows a quantitative in vivo investigation of structure and function, which can be used to investigate the pathological conditions or to evaluate the potential of novel therapeutics. A great benefit of the presented techniques is the possibility to perform longitudinal analyses allowing the investigation of dynamic processes, reducing variability and cuts down the number of animals needed for the experiments. The protocol described aims to provide a manual for acquisition and analysis of high quality retinal scans of mice and rats using a low cost customized holder with an option to deliver inhalational anesthesia. Additionally, the proposed guide is intended as an instructional manual for researchers using optokinetic response (OKR) analysis in rodents, which can be adapted to their specific needs and interests.

A detailed protocol for the assessment of structural and visual readouts in rodents by optical coherence tomography and optokinetic response is presented. The results provide valuable insights for ophthalmologic as well as neurologic research.

Optical coherence tomography (OCT) is a fast, non-invasive, interferometric technique allowing high-resolution retinal imaging. It is an ideal tool for ...

Flow Cytometric Analysis of Multiple Mitochondrial Parameters in Human Induced Pluripotent Stem Cells and Their Neural and Glial Derivatives

Mitochondria are important in the pathophysiology of many neurodegenerative diseases. Changes in mitochondrial volume, mitochondrial membrane potential (MMP), mitochondrial production of reactive oxygen species (ROS), and mitochondrial DNA (mtDNA) copy number are often features of these processes. This report details a novel flow cytometry-based approach to measure multiple mitochondrial parameters in different cell types, including human induced pluripotent stem cells (iPSCs) and iPSC-derived neural and glial cells. This flow-based strategy uses live cells to measure mitochondrial volume, MMP, and ROS levels, as well as fixed cells to estimate components of the mitochondrial respiratory chain (MRC) and mtDNA-associated proteins such as mitochondrial transcription factor A (TFAM). 
By co-staining with fluorescent reporters, including MitoTracker Green (MTG), tetramethylrhodamine ethyl ester (TMRE), and MitoSox Red, changes in mitochondrial volume, MMP, and mitochondrial ROS can be quantified and related to mitochondrial content. Double staining with antibodies against MRC complex subunits and translocase of outer mitochondrial membrane 20 (TOMM20) permits the assessment of MRC subunit expression. As the amount of TFAM is proportional to mtDNA copy number, the measurement of TFAM per TOMM20 gives an indirect measurement of mtDNA per mitochondrial volume. The entire protocol can be carried out within 2-3 h. Importantly, these protocols allow the measurement of mitochondrial parameters, both at the total level and the specific level per mitochondrial volume, using flow cytometry.

This study reports a novel approach to measure multiple mitochondrial functional parameters based on flow cytometry and double staining with two fluorescent reporters or antibodies to detect changes in mitochondrial volume, mitochondrial membrane potential, reactive oxygen species level, mitochondrial respiratory chain composition, and mitochondrial DNA.

Mitochondria are important in the pathophysiology of many neurodegenerative diseases. Changes in mitochondrial volume, mitochondrial membrane potential ...

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 ...