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

Summary

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.

Abstract

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, β-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 Müller 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.

Introduction

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 Müller glial cells (MGCs) and astrocytes, leading to adequate phototransduction and processing of visual information^1,2. 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 others^3,4,5.

On the other hand, inflammation, oxidative and nitrosative stress are involved in the pathogenesis and progression of many human diseases, including retinopathies^{6,7,8,9,10,11}. 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 (O_2͘͘͘͘^•−), hydroxyl radicals (^•OH), various peroxides (ROOR′), 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 conditions^14,15,16. 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 RD^{17,18,19,20,21,22,23,24}. 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′,7′-dichlorofluorescein diacetate (DCFH-DA) probe is one of the most widely used techniques for directly quantifying the redox state of a cell^25,26,27,28. 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′,7′-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 H₂DCF^25,29. 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.

Protocol

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 Müller glial cell line (Moorfield's/Institute of Ophthalmology- Müller 1). Always use proper aseptic technique and work in a laminar flow hood.

1. Prepare Dulbecco's modified Eagle'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.
2. Thaw the frozen MIO-M1 cells on day 1.
   1. 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 °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.
   2. Thaw the MIO-M1 cells rapidly (in less than 1 min) by warming the vial in the 37 °C water bath until there is just a small bit of ice left in the vial.
   3. Wipe the outside of the vial with 70% ethanol and transfer it to a laminar flow hood.
   4. 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.
   5. 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.
   6. 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 °C, 5% CO₂.
3. When the MIO-M1 cells become 80%-90% confluent on day 4, perform the following steps.
   1. 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.
   2. Dispense 1 mL of 0.5% Trypsin-EDTA solution and incubate at 37 °C in the CO₂ incubator for 3-5 min to detach the cells.
   3. 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.
   4. 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.
   5. Repeat the action with another 100 mm plate. Incubate the plate for approximately 1 day at 37 °C in a 5% CO₂ incubator.
4. 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.
5. 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.
   1. Put the glass cover on the Neubauer chamber central area. Place the chamber on a flat surface (e.g., a table or workbench).
   2. Mix an equal volume of trypan blue stain with the cells. For instance, mix 60 µL of trypan blue with 60 µL of cells for a 1:2 dilution.
   3. From this mixture, load 10 µL with a micropipette into a Neubauer chamber.
   4. Place the loaded Neubauer chamber on the microscope stage. Then, turn on the light and adjust the brightness.
   5. Move the microscope stage to an optimal position and adjust the focus until a sharp image of the cells is obtained.
   6. Count the cells inside the four squares (subdivided in 16) located at the corner of the hemocytometer (volume: 0.1 mm³ each ).
   7. 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.
      ​Concentration = (Number of cells x 2500 x 2) = (Number of cells x 5000 cells/mL)
   8. 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 cells/well). Shake the plate gently to distribute the cells homogeneously. Incubate the 6-well plate overnight at 37 °C in a 5% CO₂ incubator.

2. Assay conditions and controls

1. Include the following experimental controls for each experiment:
   1. Autofluorescence control (cells without DCFH-DA probe, control for setting the flow cytometer parameters).
   2. Basal control (unstimulated cells).
   3. Positive control (cells treated with a ROS inducer, A or B).
   4. Negative control (cells treated with antioxidant compound)
   5. Sample (cells treated with antioxidant compound and ROS inducer, A or B).

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.

1. 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.
2. 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 °C, 5% CO₂. This is done to starve the cells.
3. After serum starving, treat the cells with the antioxidant compound for 6 h.
   1. 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).
   2. Incubate the 6-well plate for 6 h at 37 °C, 5% CO₂.
      NOTE: If using another antioxidant compound or ROS inhibitor, standardize the optimum time and concentration for the stimuli.
4. After 6 h of incubation, treat the cells with ROS inducer, A or B, for 30 min.
   1. 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.
   2. Incubate the 6-well plate for 30 min at 37 °C, 5% CO₂.
      NOTE: If using another ROS inducer, properly standardize the optimum time and concentration for the stimuli.
5. Load the cells with the DCFH-DA probe.
   1. Aspirate the supernatant and wash the cells in the 6-well plate 3x with PBS to remove all the stimuli.
   2. 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 µM DCFH-DA in DMEM without phenol red in a 15 mL tube.
   3. 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).
   4. Add 1 mL of DMEM without phenol red to the autofluorescence control cells.
   5. Incubate the 6-well plate for 30 min at 37 °C, 5% CO₂.
      ​NOTE: The probe is light sensitive. Discard all the unused probe solution.

4. Cell preparation for flow cytometry

1. 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.
2. 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.
3. Collect them in labeled 1.5 mL tubes and centrifuge at 600 x g for 5 min at 4 °C.
   NOTE: When harvesting the cells, avoid bubble formation by setting the P1000 pipette to 0.4 mL. From this step onward, work fast.
4. 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.
5. 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 °C. Repeat these steps 2x more. (Perform three washes in total).
6. When the last wash ends, label 5 mL round-bottom polystyrene tubes (flow cytometry tubes) for all experimental conditions (see step 2.).
7. When centrifugation ends, resuspend the cell pellets in 200 µL 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.

5. Data acquisition in a flow cytometer

1. 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).
2. 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.
3. 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.).
4. 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.
5. Click the Current Tube Pointer icon to select the tube, and the icon will change to green. Place the "autofluorescence control" tube on the aspirator arm, moving the base carefully only to the left.
   1. To run the "autofluorescence control" 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).
   2. 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.
6. To set the FITC voltage, run the "positive control (cells treated with a ROS inducer, A or B)". 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.
7. Place again the "autofluorescence control" 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.
8. 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.
   1. 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.
9. For recording the next samples, remove the tube and click Next tube > Acquire Data > Record Data.
   NOTE: The flow cytometer used here (see Table of Materials) records a maximum of 1 x 10⁶ events per tube.
10. When the recording of all tubes ends, export the data to disk D. To do this click File > Export > FCS files > 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.

6. Analysis of the acquired data

1. Open the software and add the samples using the Add Samples action button; it is located on the taskbar or in the Navigate band.
   1. Click Add Samples.
   2. Using the file browser, navigate to the Experimental folder.
   3. Select the Experimental folder and click Choose.
      NOTE: The sample files load as part of the "All Samples" group into the workspace.
2. 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.
3. Draw a polygon gate to isolate the cells of interest, excluding dead cells and debris.
   1. Click the Polygon Gate tool.
   2. Click within the plot to make up the polygon gate. Make as many sides as needed.
   3. Double-click at the last point to close the polygon and create the gate desired.
   4. Provide a name for the gate, such as "MIO-M1 cells", and press OK.
      ​NOTE: A new graph plot window appears displaying only the MIO-M1 events without dead cells and debris.
   5. Repeat with all the samples to analyze.
4. 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.
5. Add Statistics using the Add Statistic Window.
   1. Below of histogram graph, click Add Statistics. The program will open a new statistic window.
   2. On the left of the new window select the statistics Geometric Mean.
   3. Select the Population MIO-M 1 cells.
   4. From the list of available parameters, select FIT-C.
   5. Click the Add button to apply them to the analysis.
      NOTE: This creates the new statistics "geometric mean: FITC" in the sample gating tree, and their values are displayed in your workspace.
6. Put these geometric mean values in a statistics program and analyze.
7. 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.
   1. From the gating tree of a sample in the Workspace window, drag MIO-M 1 cells population into the Layout Editor.
   2. 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.
   3. Drag all the samples to compare them in the same graph. The program will overlap them.
   4. 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.
   5. Right-click the sample and change Coloring, Line Style, Line Weight, etc. to personalize the graph.
   6. 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.
8. Save the analysis as a workspace (.wsp) File.
   1. On the top-left corner, click the Cytometry Analysis icon and select Save As.
   2. Choose Save as Workspace (WSP) to save the document with data and analysis.
   3. Enter a name for the workspace file.
   4. Click Save.

Results

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 "basal control" vs. "autofluorescence control", dot plots graph). This occurred due to a basal production of ROS in the MIO-M1 cells....

Discussion

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 overproduction^{6,7,8,9,10,11}. Therefore, the detection, measurement, and understanding of the pathway involved in the modul...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
2′,7′-DCFH-DA	Sigma	35845-1G
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)	Gibco by life technologies	15630-080
BD FACSCanto II flow cytometer	BD Biosciences	FACSCanto
BD FACSDiva software	BD Biosciences
Cell Culture Dishes 100x20 mm	Cell Star- Greiner Bio-One	664 160
Centrifuge	Thermo	Sorvall legend micro 17 R
Centrifuge Tubes (15 ml)	BIOFIL	CFT011150
Centrifuge Tubes (50 ml)	BIOFIL	CFT011500
Cryovial	CRYO.S - Greiner Bio-One	126263
Dimethyl Sulfoxide	Sigma-Aldrich	W387520-1KG
Disodium-hydrogen-phosphate heptahydrate	Merck	106575
DMEM without phenol red	Gibco by life technologies	31053-028
Dulbecco’s modified Eagle’s medium (DMEM)	Gibco by life technologies	11995065
Ethylenediamine Tetraacetic Acid (EDTA), Disodium Salt, Dihydrate	Merck	324503
Fetal Bovine Serum	Internegocios
FlowJo v10 Software	BD Biosciences
Glucose	Merck	108337
hemocytometer, Neubauer chamber	BOECO,Germany
Laminar flow hood	ESCO	AC2-6E8
L-glutamine (GlutaMAX)	Gibco by life technologies	A12860-01
MitoSOX Red	Invitrogen	M36008
Penicillin/Streptomycin	Gibco by life technologies	15140-122
Potassium Chloride	Merck	104936
Potassium-dihydrogen phosphate	Merck	4878
Round polystyrene tubes 5 ml (flow cytometry tubes)	Falcon - Corning	BD-352008
Sodium Azide	Merck	822335
Sodium Chloride	Merck	106404
Sodium Hydroxide	Merck	106462
SPINWIN Micro Centrifuge Tube 1.5 ml	Tarson	500010-N
Tissue Culture Plate 6 well	BIOFIL	TCP011006
Trypan Blue	Merck	111732
Trypsin-EDTA 0.5% 10X	Gibco by life technologies	15400-054
Vortex Mixer	Labnet International, Inc.