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.
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.
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 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 (O2͘͘͘͘•−), 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 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′,7′-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′,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 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.
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.
2. Assay conditions and controls
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.
4. Cell preparation for flow cytometry
5. Data acquisition in a flow cytometer
6. Analysis of the acquired data
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. (Figure 1B, compare "basal control" vs. "autofluorescence control", 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 "ROS inducer A" samples vs. "basal control" and "ROS inducer B" samples vs. "basal control", 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 "ROS inducer A" vs. "pretreatment with antioxidant compound 6 h + ROS inducer A 30 min", and "ROS inducer B" vs. "pretreatment with antioxidant compound 6 h + ROS inducer B 30 min", 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 "6h antioxidant compound" vs. "basal control").
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).
Figure 1: Measurement of ROS in response to ROS inducer, A or B, using DCFH-DA probe in MIO-M1 cells: (A) Comparison of "basal control" vs. "autofluorescence control" 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 ± SEM and analyzed by one-way ANOVA followed by Dunnett's post-test; **p < 0.01, ***p < 0.001. Please click here to view a larger version of this figure.
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 ± SEM and analyzed by one-way ANOVA followed by Dunnett's post-test; ns, not significant, **p < 0.01, ***p < 0.001. Please click here to view a larger version of this figure.
Solution | Storage conditions | Note | |||||
Sodium Hydroxide (NaOH) 10 M, 1 L | 400 g NaOH pellets | distilled water to 1 L | RT | “CAUTION”: 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. | |||
Phosphate-Buffered Saline (PBS) 10 X, 1 L | 80 g NaCl | distilled water to 1 L | RT | Adjust pH to 7.4 and filter the solution. | |||
2 g KCl | |||||||
11.5 g Na2HPO4· 7H2O | |||||||
2 g KH2PO4 | |||||||
Ethylenediamine Tetraacetic Acid (EDTA), 0.5 M pH 8.0, 1 L | 186.1 g Na2EDTA· 2H2O | distilled water to 1 L | RT | 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. | |||
10% w/v Sodium Azide (NaN3), 100 mL | 10 g NaN3 | distilled water to 100 mL | RT | ||||
Detaching Buffer, 100 mL | 180 mg glucose | PBS 1X to 100 mL | 4 °C | ||||
0.6 mL 0.5 M pH 8.0 EDTA | |||||||
Flow Cytometry Staining Buffer (FACS Buffer), 100 mL | 2 mL fetal bovine serum (FBS) | PBS 1X to 100 mL | 4 °C | NaN3 is added as a preservative. | |||
1 mL 0.5 M pH 8.0 EDTA | |||||||
1 mL 10% w/v sodium azide | |||||||
5 mM 2′,7′-DCFH-DA, 1 mL | 2.4 mg DCFH-DA | dimethyl sulfoxide to 1 mL | -20 °C protected from light | Mix gently and aliquot 20 μL in 1.5 mL tubes. Avoid multiple thaw/freeze cycles. Our recommendation is discarding all unused probe solution from the experimental day. | |||
0.4% w/v trypan blue 10 X, 50 mL | 0.2 g trypan blue | PBS 1X to 50 mL | 4 °C |
Table 1: Buffer recipes
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°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. 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 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 µM, 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 5µM and 10 µM, 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 authors have nothing to disclose.
The authors would like to thank María Pilar Crespo and Paula Alejandra Abadie of CIBICI (Centro de Investigaciones en Bioquímica Clínica e Inmunología, CONICET-UNC, Córdoba, 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 Secretaría de Ciencia y Tecnología, Universidad Nacional de Córdoba (SECyT-UNC) Consolidar 2018-2021, Fondo para la Investigación Científica y Tecnológica (FONCyT), and Proyecto de Investigación en Ciencia y Tecnología (PICT) 2015 N° 1314 (all to M.C.S.).
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 100×20 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. |