Abstract
Cells constantly generate reactive oxygen species (ROS) during aerobic metabolism. The ROS generation plays an important protective and functional role in the immune system. The cell is armed with a powerful antioxidant defense system to combat excessive production of ROS. Oxidative stress occurs in cells when the generation of ROS overwhelms the cells’ natural antioxidant defenses. ROS and the oxidative damage are thought to play an important role in many human diseases including cancer, atherosclerosis, other neurodegenerative diseases and diabetes. Thus, establishing their precise role requires the ability to measure ROS accurately and the oxidative damage that they cause. There are many methods for measuring free radical production in cells. The most straightforward techniques use cell permeable fluorescent and chemiluminescent probes. 2′-7′-Dichlorodihydrofluorescein diacetate (DCFH-DA) is one of the most widely used techniques for directly measuring the redox state of a cell. It has several advantages over other techniques developed. It is very easy to use, extremely sensitive to changes in the redox state of a cell, inexpensive and can be used to follow changes in ROS over time.
Key words
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsReferences
Halliwell B, Gutteridge JM (1999) Free radicals in biology and medicine. Oxford University Press, Oxford
Sohal RS, Mockett RJ, Orr WC (2002) Mechanisms of aging: an appraisal of the oxidative stress hypothesis. Free Radic Biol Med 33:575–586
Halliwell B (2001) Role of free radicals in the neurodegenerative diseases: therapeutic implications for antioxidant treatment. Drugs Aging 18:685–716
Butterfield DA (2002) Amyloid beta-peptide (1–42)-induced oxidative stress and neurotoxicity: implications for neurodegeneration in Alzheimer’s disease brain. Rev Free Radic Res 36:1307–1313
Liu Q, Raina AK, Smith MA, Sayre LM, Perry G (2003) Hydroxynonenal, toxic carbonyls, and Alzheimer disease. Mol Aspects Med 24:305–313
Hagen TM, Huang S, Curnutte J, Flower P, Martinez V, Wehr CM, Ames BN, Chisari F (1994) Extensive oxidative DNA damage in hepatocytes of transgenic mice with chronic active hepatitis destined to develop hepatocellular carcinoma. Proc Natl Acad Sci U S A 91:12808–12812
Chowienczyk PJ, Brett SE, Gopaul NK, Meeking D, Marchetti M, Russel-Jones DL, Anggard EE, Ritter JM (2000) Oral treatment with an antioxidant (raxofelast) reduces oxidative stress and improves endothelial function in men with type II diabetes. Diabetologia 43:974–977
Parthasarathy S, Santanam N, Ramachandran S, Meilhac O (2000) Potential role of oxidized lipids and lipoproteins in antioxidant defense. Free Radic Res 33:197–215
Beyer RE (1992) An analysis of the role of coenzyme Q in free radical generation and as an antioxidant. Biochem Cell Biol 70:390
Raha S, McEachern GE, Myint AT, Robinson BH (2000) Superoxides from mitochondrial complex III: the role of manganese superoxide dismutase. Free Radic Biol Med 29:170
Finkel T (2000) Redox-dependent signal transduction. FEBS Lett 476:52
Tammariello SP, Quinn MT, Estus S (2000) NADPH oxidase contributes directly to oxidative stress and apoptosis in nerve growth factor-deprived sympathetic neurons. J Neurosci 20:RC53
Suzuki Y, Ono Y, Hirabayashi Y (1998) Rapid and specific reactive oxygen species generation via NADPH oxidase activation during Fas-mediated apoptosis. FEBS Lett 425:209
Hampton MB, Orrenius S (1997) Dual regulation of caspase activity by hydrogen peroxide: implications for apoptosis. FEBS Lett 414:552
Creagh EM, Cotter TG (1999) Selective protection by hsp 70 against cytotoxic drug-, but not Fas-induced T-cell apoptosis. Immunology 97:36
Michiels C, Raes M, Toussaint O, Remacle J (1994) Importance of Se-glutathione peroxidase, catalase, and Cu/Zn-SOD for cell survival against oxidative stress. Free Radic Biol Med 17:235
Reed DJ (1990) Glutathione: toxicological implications. Annu Rev Pharmacol Toxicol 30:603
Lledias F, Rangel P, Hansberg W (1998) Oxidation of catalase by singlet oxygen. J Biol Chem 273:10630
Bai J, Rodriguez AM, Melendez JA, Cederbaum AI (1999) Overexpression of catalase in cytosolic or mitochondrial compartment protects HepG2 cells against oxidative injury. J Biol Chem 274:26217
Murphy TH, De Long MJ, Coyle JT (1991) Enhanced NAD(P)H:quinone reductase activity prevents glutamate toxicity produced by oxidative stress. J Neurochem 56:990
Mustacich D, Powis G (2000) Thioredoxin reductase. Biochem J 346:1–8
Kasahara Y, Iwai K, Yachie A, Ohta K, Konno A, Seki H, Miyawaki T, Taniguchi N (1997) Involvement of reactive oxygen intermediates in spontaneous and CD95 (Fas/APO-1)-mediated apoptosis of neutrophils. Blood 89:1748
Lundqvist-Gustafsson H, Bengtsson T (1999) Activation of the granule pool of the NADPH oxidase accelerates apoptosis in human neutrophils. J Leukoc Biol 65:196
Oishi K, Machida K (1997) Inhibition of neutrophil apoptosis by antioxidants in culture medium. Scand J Immunol 45:21
Woo CH, Eom YW, Yoo MH, You HJ, Han HJ, Song WK, Yoo YJ, Chun JS, Kim JH (2000) Tumor necrosis factor-alpha generates reactive oxygen species via a cytosolic phospholipase A2-linked cascade. J Biol Chem 275:32357–32362
Elchuri S, Oberley TD, Qi W, Eisenstein RS, Jackson Roberts L, Van Remmen H, Epstein CJ, Huang TT (2005) CuZnSOD deficiency leads to persistent and widespread oxidative damage and hepatocarcinogenesis later in life. Oncogene 24:367–380
Li Y, Huang TT, Carlson EJ, Melov S, Ursell PC, Olson JL, Noble LJ, Yoshimura MP, Berger C, Chan PH et al (1995) Dilated cardiomyopathy and neonatal lethality in mutant mice lacking manganese superoxide dismutase. Nat Genet 11:376–381
Evans MD, Dizdaroglu M, Cooke MS (2004) Oxidative DNA damage and disease: induction, repair and significance. Mutat Res 567:1–61
Kawanishi S, Hiraku Y, Pinlaor S, Ma N (2006) Oxidative and nitrative DNA damage in animals and patients with inflammatory diseases in relation to inflammation-related carcinogenesis. Biol Chem 387:365–372
Mori K, Shibanuma M, Nose K (2004) Invasive potential induced under long-term oxidative stress in mammary epithelial cells. Cancer Res 64:7464–7472
Radisky DC, Levy DD, Littlepage LE, Liu H, Nelson CM, Fata JE, Leake D, Godden EL, Albertson DG, Nieto MA et al (2005) Rac1b and reactive oxygen species mediate MMP-3-induced EMT and genomic instability. Nature 436:123–127
Kusmartsev S, Nefedova Y, Yoder D, Gabrilovich DI (2004) Antigen-specific inhibition of CD8+ T cell response by immature myeloid cells in cancer is mediated by reactive oxygen species. J Immunol 172:989–999
Corzo CA, Nagaraj S, Kusmartsev S, Gabrilovich D (2007) Role of reactive oxygen species in immune suppression in cancer. J Immunol 178:49.12
Kusmartsev S, Eruslanov E, Kübler H, Tseng T, Sakai Y, Su Z, Kaliberov S, Heiser A, Rosser C, Dahm P, Siemann D, Vieweg J (2008) Oxidative stress regulates expression of VEGFR1 in myeloid cells: Link to tumor-induced immune suppression in renal cell carcinoma. J Immunol 181:346–353
Dahlgren C, Karlsson A (1999) Respiratory burst in human neutrophils. J Immunol Methods 232:3
Mellqvist UH, Hansson M, Brune M, Dahlgren C, Hermodsson S, Hellstrand K (2000) Natural killer cell dysfunction and apoptosis induced by chronic myelogenous leukemia cells: role of reactive oxygen species and regulation by histamine. Blood 96:1961
Gyllenhammar H (1987) Lucigenin chemiluminescence in the assessment of neutrophil superoxide production. J Immunol Methods 97:209
Nourooz-Zadeh J (1999) Ferrous ion oxidation in presence of xylenol orange for detection of lipid hydroperoxides in plasma. Methods Enzymol 300:58
Ottonello L, Frumento G, Arduino N, Dapino P, Tortolina G, Dallegri F (2001) Immune complex stimulation of neutrophil apoptosis: investigating the involvement of oxidative and nonoxidative pathways. Free Radic Biol Med 30:161–169
Bass DA, Parce JW, Dechatelet LR, Szejda P, Seeds MC, Thomas M (1983) Flow cytometric studies of oxidative product formation by neutrophils: a graded response to membrane stimulation. J Immunol 130:1910
Royall JA, Ischiropoulos H (1993) Evaluation of 2′,7′-dichlorofluorescin and dihydrorhodamine 123 as fluorescent probes for intracellular H2O2 in cultured endothelial cells. Arch Biochem Biophys 302:348–355
Ubezio P, Civoli F (1994) Flow cytometric detection of hydrogen peroxide production induced by doxorubicin in cancer cells. Free Radic Biol Med 16:509
Halliwell B, Whitemann M (2004) Measuring reactive species and oxidative damage in vivo and in cell culture:how should you do it and do the results mean? Br J Pharmacol 142:231–255
Ischiropoulos H, Gow A, Thom SR, Kooy NM, Royall JA, Crow JP (1999) Detection of reactive nitrogen species using 2, 7-dichlorodihydrofluorescein and dihydrorhodamine 123. Methods Enzymol 301:367–373
Bilski P, Belanger AG, Chignell CF (2002) Photosensitized oxidation of 2’, 7’-dichlorofluorescin: singlet oxygen does not contribute to the formation of fluorescent oxidation product 2’, 7’-dichlorofluorescein. Free Radic Biol Med 33:938–946
Ohashi T, Mizutani A, Mukarima A, Kojo S, Ishii T, 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 511:21–27
Tampo Y, Kotamraju S, Chitambar RC, Kalivendi SV, Kezler A, Joesph J, Kalyanaraman B (2003) Oxidative stress-induced iron signaling is responsible for peroxide-dependent oxidation of dichlorodihydrofluorescein in endotheliel cells. Role of transferrin receptor-dependent iron uptake in apoptosis. Circ Res 92:56–63
Lebel CP, Ischiropoulos H, Bondy SC (1992) Evaluation of the probe 2’, 7’-dichlorofluorescin as an indicator of reactive oxygen species formation and oxidative stress. Chem Res Toxicol 5:227–231
Rao KM, Padmanabhan J, Kilby DL, Cohen HJ, Currie MS, Weinberg JB (1992) Flow cytometric analysis of nitric oxide production in human neutrophils using dichlorofluorescein diacetate in the presence of a calmodulin inhibitor. J Leukoc Biol 51:496
Kooy NW, Lewis SJ, Royall JA, Ye YZ, Kelly DR, Beckman JS (1997) Extensive tyrosine nitration in human myocardial inflammation: evidence for the presence of peroxynitrite. Crit Care Med 25:812–819
Rota C, Fann YC, Mason RP (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 274:28161–28168
Lawrence A, Jones CM, Wardman P, Burkitt MJ (2003) Evidence for the role of a peroxidase compound I-type intermediate in the oxidation of glutathione, NADH, ascorbate, and dichlorofluorescin by cytochrome c/H2O2. Implications for oxidative stress during apoptosis. J Biol Chem 278:29410–29419
Martin BD, Schoenhard JA, Sugden KD (1998) Hypervalent chromium mimics reactive oxygen species as measured by the oxidant-sensitive dyes 2′, 7′-dichlorofluorescin and dihydrorhodamine. Chem Res Toxicol 11:1402–1410
O’Malley YQ, Reszka KJ, Britigan BE (2004) Direct oxidation of 2′, 7′-dichlorodihydrofluorescein by pyocyanin and other redox-active compounds independent of reactive oxygen species production. Free Radic Biol Med 36:90–100
Henzler T, Steudle E (2000) Transport and metabolic degradation of hydrogen peroxide in Chara corallina: model calculations and measurements with the pressure probe suggest transport of H(2)O(2) across water channels. J Exp Bot 51:2053–2066
Marla SS, Lee J, Groves JT (1997) Peroxynitrite rapidly permeates phospholipid membranes. Proc Natl Acad Sci U S A 94:14243–14248
Clement MV, Long LH, Ramalingam J, Halliwell B (2002) The cytotoxicity of dopamine may be an artefact of cell culture. J Neurochem 81:414–421
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2010 Humana Press, a part of Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Eruslanov, E., Kusmartsev, S. (2010). Identification of ROS Using Oxidized DCFDA and Flow-Cytometry. In: Armstrong, D. (eds) Advanced Protocols in Oxidative Stress II. Methods in Molecular Biology, vol 594. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60761-411-1_4
Download citation
DOI: https://doi.org/10.1007/978-1-60761-411-1_4
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-60761-410-4
Online ISBN: 978-1-60761-411-1
eBook Packages: Springer Protocols