Measurement of Reactive Oxygen and Nitrogen Species in Living Cells Using the Probe 2',7'-Dichlorodihydrofluorescein

Materials and Reagents

1. CM-DCFH₂-DA (Invitrogen Life Technologies/Molecular Probes, EUA), storage temperature at -20°C

2. High quality anhydrous dimethylsulfoxide (DMSO), dimethyl-formamide (DMF) or 100% ethanol, H₂O₂, and organic salts (Merck, Brazil), storage in 15/30°C

3. Loading buffer, such as a simple physiological buffer (PBS), storage in 4°C fridge

4. N-acetyl-L-cysteine (NAC) (Sigma, catalog number: A7250), storage in 4°C fridge

5. Buthionine Sulfoximine (BSO) (Sigma, catalog number: B2515), storage in 4°C fridge

6. HUVEC were obtained from the American Type Culture Collection, cultivated in RPMI 1640 medium supplemented with 10% fetal bovine serum, penicillin, and streptomycin antibiotics. Experiments were performed at 37°C and in a humidified atmosphere of 95% air and 5% CO₂.

7. PBS (see Recipes)

8. Phosphate buffer (see Recipes), storage in 4°C fridge

Equipment

1. Spectrophotometer model U-2010 (Software UV Solutions, version 1.1, Hitachi, Japan)

2. 96-well microplates (black-walled, clear bottom; Corning, catalog number: CLS3829)

Software

1. GraphPad Prism Version 5.01 (or higher) (GraphPad Software Inc.)

Procedure

1. Remove growth media from adherent or suspended cells and then wash twice with PBS 1×. Balance to 37°C before use. The 1× Buffer can be kept frozen or at 4°C for future use.

2. The medium should not contain phenol red or other colorimetric dyes, before and during the assay. The CM-DCFH₂-DA should only be dissolved in anhydrous DMSO, DMF, or ethanol (100%). The working solutions must be freshly prepared in a light-protected vial, and tightly closed until use.

3. For a standard assay used often, dissolve 50 mg of CM-DCFH₂-DA (MW 487.3) in 2.565 ml of DMSO or absolute ethanol (40 mM). Aliquots can be kept at -20°C for at least 3 months. Dilute 100 µl of the stock solution with 39.9 ml of pre-warmed culture medium (final working concentration 100 μM), vortex, and add to the cell culture wells.

4. CM-DCFH₂-DA should be utilized in concentrations as low as possible to decrease potential artifacts, such as incomplete hydrolysis, and contamination by hydrolytic by-products. The ideal working solution for each application must be determined empirically, to obtain a good signal-to-noise ratio, e.g., by 2-3 further 2-fold dilutions of the standard concentration.

5. Incubate the cells in the Ultra-Low Cluster Plate (specific black coated 96-well dish) in the cell incubator (37°C), with high relative humidity (95%), and a controlled level of carbon dioxide (5%) in the dark. Usually, a loading time of 2 h is sufficient. Additionally, if multi-well plates and an automated plate reader are used, it is crucial to estimate the total amount of cells, cell density, and/or total protein level for each individual well, to allow normalization and secure optimal cell density and viability.

6. Wash carefully with the cell buffer, followed by incubation, allowing a brief recovery period for cellular esterases to hydrolyze the acetate groups. Let the dye respond to oxidation.

7. A suggested positive buffer control is H₂O₂, or tert-Butyl hydroperoxide (TBHP) at a final concentration of 50 μM.

8. The redox state of the sample can be measured via the increasing fluorescence of DCF in cells, as measured at 530 nm when the sample is excited at 485 nm.

9. Importantly, the fluorescence of negative controls without DCFH₂-DA maintained in loading buffer needs to be determined as background.

10. In most healthy cells, oxygen radicals are eliminated by cellular enzymes and/or natural antioxidants, resulting in a usually low level of fluorescence.

11. Power up the fluorescence plate reader at a suitable interval prior to the initiation of the time course measurements. Describe the plate dimension situations and choose the appropriate filter set for excitation/detection of fluorescence.

12. Read the fluorescence on each plate and print the plate data or save in digital form.

13. Average the interpretations from the triplicates for each experimental condition and calculate the percent coefficient of variation (CV) for each of the means.

14. Subtract the fluorescence average of the cell-free treatment control from that of the condition treated under the equivalent experimental conditions to determine the intracellular DCF fluorescence. Calculate the mean fluorescence and CV for each triplicate, confirming that the triplicates are consistent.

Data analysis

Techniques for measuring RONS with the DCFH₂-DA/DCFH₂ system have certain weaknesses, since all the assays are prone to numerous artifacts, resulting from sample preparation or from the analytical method itself. Organic solvents, such as DMSO or ethanol, which are used to dissolve the test compounds, are another difficulty that all tests have in common. DMSO and ethanol are powerful •OH scavengers, which—although they are highly diluted in the assay and mainly extracellular—could lead to an underestimation of the amount of RONS produced. Therefore, appropriate controls are very important, because the results can be altered by various reactions taking place in the culture medium, or by handling of the cell cultures that may lead to oxidative stress (Tarpey et al., 2004). RONS measurements in biological systems do not provide absolute quantification of RONS levels, as discussed below (Figure 3).

Notes

1. DCFH₂ has little or no reactivity with O₂•^–

   1. O₂•^–, produced by the xanthine/XO system, is quickly inhibited by superoxide dismutase, which prevents detection by oxidation of DCFH₂ (LeBel et al., 1992).

   2. Reduction of cytochrome c induced by potassium O₂•^– does not rise DCF fluorescence, indicating that O₂•^– does not directly oxidize DCFH₂ (Zhu et al., 1994).

   3. A steady-state radiolysis providing a continuous supply of O₂•^– also does not cause significant oxidation of DCFH₂ (Zhu et al., 1994; Wrona et al., 2005).

2. H₂O₂ cannot directly oxidize DCFH₂

   1. H₂O₂-dependent oxidation of DCFH₂ to DCF occurs slowly, if at all, in the absence of ferrous iron. However, DCF formation is greatly enhanced in the presence of heme-containing substances, such as hematin, peroxidases, cytochrome c, or other redox-active metal ions (Cathcart et al., 1984; Kalyanaraman et al., 2012).

   2. Peroxidases are capable of inducing DCFH₂ oxidation in the absence of H₂O₂ (Rota et al., 1999). Therefore, increases in cellular peroxidase or heme activity are likely to be equally, if not more important than H₂O₂, in determining rates of DCF formation and cellular fluorescence (Ohashi et al., 2002).

   3. Enhanced intracellular iron trafficking, such as transferrin receptor resulting in increased iron uptake into endothelial cells, usually promotes H₂O₂-dependent DCF fluorescence (Tampo et al., 2003).

3. Oxidation of DCFH₂ by the •OH radical

   1. For the Fe²⁺/H₂O₂ system (Fenton reaction), •OH scavengers such as DMSO, ethanol, mannitol, and Tris only partially inhibit DCFH₂ oxidation (LeBel et al., 1992).

   2. Free •OH, which is very short lived (<20 ms in a typical physiological environment), is not involved in the oxidation of DCFH₂, while site-specific •OH formed by the binding of the DCFH₂ carboxyl group to Fe²⁺ can oxidize DCFH₂. Thus, Fe²⁺/H₂O₂–induced •OH generation (indicated by the site specific oxidation of salicylic acid to 2,3-dihydroxybenzoic acid), which is proportional to DCFH₂ oxidation, can be repressed by DMSO and mannitol (Zhu et al., 1994).

   3. DMSO has been observed to inhibit DCFH₂ oxidation in several cells or cell lines, where •OH has also been shown to oxidize DCFH₂ with high efficacy (Wrona et al., 2005).

4. DCFH₂ as an excellent indicator of ONOO^– formation in living cells

   1. ONOO^–-mediated oxidation of DCFH₂ is much more rapid and efficient than oxidation by H₂O₂, NO, or O₂•^–. ONOO^– reacting with DCFH₂ reached a steady-state after 2 min, whereas H₂O₂ and NO continued to oxidize DCFH₂ for at least 10 min (Possel et al., 1997).

   2. In vitro oxidation of DCFH₂ mediated by ONOO^– was not inhibited by •OH scavengers, and was not dependent upon metal ion-catalyzed reactions. DCFH₂-DA was not susceptible to oxidation by ONOO^–, while ONOO^– could readily oxidize DCFH₂ (Crow, 1997). The lack of an inhibitory effect by the •OH scavenger mannitol and DMSO indicates that the oxidation reaction of ONOO^– with DCFH₂ is not based on the formation of free H₂O₂, which rapidly forms from the combination of two •OH radicals.

   3. Metal catalysis is not required for efficient ONOO^–-mediated DCFH₂ oxidation. This reaction appears to be mediated directly by ONOO^–, and does not require the secondary formation of other free radicals (Kooy et al., 1997).

   4. HKGreen-1 possesses a ketone unit linked to a DCF moiety through an aryl ether linkage and provided a highly selective fluorescent probe for the detection of ONOO^– in primary cultured neuronal cells (Yang et al., 2006).

Recipes

1. PBS

   10× Buffer. Prepare 1× Buffer by diluting 10× buffer in deionized water to make 100 ml 1× Buffer. Combine 10 ml 10× Buffer with 90 ml deionized water and mixture gently. Marker this solution as “1× Buffer.”

2. Phosphate buffer (10 mM, pH 7.4) to lyse cells

   5 ml of cold absolute methanol (v/v)

   10 µl of Triton X100

   5 ml of Buffer A + B

   A: 9.5 ml 200 mM KH₂PO₄

   B: 40.5 ml 200 mM K₂HPO₄

Acknowledgments

DS receives project-related support from the EU Horizon 2020 under grant agreement n. 634413 (EPoS, European Project on Steatohepatitis) and 777377 (LITMUS, Liver Investigation on Marker Utility in Steatohepatitis), and by the German Research Foundation collaborative research project grants DFG CRC 1066/B3 and CRC 1292/B8.

Competing interests

The authors declare they have no competing interests.

References

1. Albrecht, S. C., Barata, A. G., Grosshans, J., Teleman, A. A. and Dick, T. P. (2011). In vivo mapping of hydrogen peroxide and oxidized glutathione reveals chemical and regional specificity of redox homeostasis. Cell Metab 14(6): 819-829.
2. Brandes, R. P., Rezende, F. and Schröder, K. (2018). Redox Regulation Beyond ROS: Why ROS Should Not Be Measured as Often. Circ Res 123(3): 326-328.
3. Cathcart, R., Schwiers, E. and Ames, B. N. (1984). Detection of picomole levels of lipid hydroperoxides using a dichlorofluorescein fluorescent assay. Methods Enzymol 105: 352-358.
4. Crow, J. P. (1997). Dichlorodihydrofluorescein and dihydrorhodamine 123 are sensitive indicators of peroxynitrite in vitro: implications for intracellular measurement of reactive nitrogen and oxygen species. Nitric Oxide 1(2): 145-157.
5. Dröge, W. (2002). Free radicals in the physiological control of cell function. Physiol Rev 82(1): 47-95.
6. Dikalov, S. I. and Harrison, D. G. (2014). Methods for detection of mitochondrial and cellular reactive oxygen species. Antioxid Redox Signal 20(2): 372-382.
7. Glebska, J. and Koppenol, W. H. (2003). Peroxynitrite-mediated oxidation of dichlorodihydrofluorescein and dihydrorhodamine. Free Radic Biol Med 35(6): 676-682.
8. Halliwell, B. and Whiteman, M. (2004). Measuring reactive species and oxidative damage in vivo and in cell culture: how should you do it and what do the results mean? Br J Pharmacol 142(2): 231-255.
9. Halliwell, B. (2012). Free radicals and antioxidants: updating a personal view. Nutr Rev 70(5): 257-265.
10. Harris, I. S. and Brugge, J. S. (2015). Cancer: The enemy of my enemy is my friend. Nature 527(7577): 170-171.
11. Hempel, S. L., Buettner, G. R., O'Malley, Y. Q., Wessels, D. A. and Flaherty, D. M. (1999). Dihydrofluorescein diacetate is superior for detecting intracellular oxidants: comparison with 2',7'-dichlorodihydrofluorescein diacetate, 5(and 6)-carboxy-2',7'-dichlorodihydrofluorescein diacetate, and dihydrorhodamine 123. Free Radic Biol Med 27(1-2): 146-159.
12. Kalyanaraman, B., Darley-Usmar, V., Davies, K. J., Dennery, P. A., Forman, H. J., Grisham, M. B., Mann, G. E., Moore, K., Roberts, L. J., 2nd and Ischiropoulos, H. (2012). Measuring reactive oxygen and nitrogen species with fluorescent probes: challenges and limitations. Free Radic Biol Med 52(1): 1-6.
13. Kooy, N. W., Royall, J. A. and Ischiropoulos, H. (1997). Oxidation of 2',7'-dichlorofluorescin by peroxynitrite. Free Radic Res 27(3): 245-254.
14. LeBel, C. P., Ischiropoulos, H. and Bondy, S. C. (1992). Evaluation of the probe 2',7'-dichlorofluorescin as an indicator of reactive oxygen species formation and oxidative stress. Chem Res Toxicol 5(2): 227-231.
15. Murphy, M. P., Holmgren, A., Larsson, N. G., Halliwell, B., Chang, C. J., Kalyanaraman, B., Rhee, S. G., Thornalley, P. J., Partridge, L., Gems, D., et al. (2011). Unraveling the biological roles of reactive oxygen species. Cell Metab 13(4): 361-366.
16. Ohashi, T., Mizutani, A., Murakami, A., Kojo, S., Ishii, T. and Taketani, S. (2002). Rapid oxidation of dichlorodihydrofluorescin with heme and hemoproteins: formation of the fluorescein is independent of the generation of reactive oxygen species. FEBS Lett 511(1-3): 21-27.
17. Possel, H., Noack, H., Augustin, W., Keilhoff, G. and Wolf, G. (1997). 2,7-Dihydrodichlorofluorescein diacetate as a fluorescent marker for peroxynitrite formation. FEBS Lett 416(2): 175-178.
18. Rota, C., Fann, Y. C. and Mason, R. P. (1999). Phenoxyl free radical formation during the oxidation of the fluorescent dye 2',7'-dichlorofluorescein by horseradish peroxidase. Possible consequences for oxidative stress measurements. J Biol Chem 274(40): 28161-28168.
19. Tampo, Y., Kotamraju, S., Chitambar, C. R., Kalivendi, S. V., Keszler, A., Joseph, J. and Kalyanaraman, B. (2003). Oxidative stress-induced iron signaling is responsible for peroxide-dependent oxidation of dichlorodihydrofluorescein in endothelial cells: role of transferrin receptor-dependent iron uptake in apoptosis. Circ Res 92(1): 56-63.
20. Tarpey, M. M., Wink, D. A. and Grisham, M. B. (2004). Methods for detection of reactive metabolites of oxygen and nitrogen: in vitro and in vivo considerations. Am J Physiol Regul Integr Comp Physiol 286(3): R431-444.
21. Winterbourn, C. C. (2008). Reconciling the chemistry and biology of reactive oxygen species. Nat Chem Biol 4(5): 278-286.
22. Wrona, M., Patel, K. and Wardman, P. (2005). Reactivity of 2',7'-dichlorodihydrofluorescein and dihydrorhodamine 123 and their oxidized forms toward carbonate, nitrogen dioxide, and hydroxyl radicals. Free Radic Biol Med 38(2): 262-270.
23. Yang, D., Wang, H. L., Sun, Z. N., Chung, N. W. and Shen, J. G. (2006). A highly selective fluorescent probe for the detection and imaging of peroxynitrite in living cells. J Am Chem Soc 128(18): 6004-6005.
24. Zhu, H., Bannenberg, G. L., Moldéus, P. and Shertzer, H. G. (1994). Oxidation pathways for the intracellular probe 2',7'-dichlorofluorescein. Arch Toxicol 68(9): 582-587.