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Simultaneous analysis of reactive oxygen species and reduced glutathione content in living cells by polychromatic flow cytometry

Nature Protocols volume 4, pages 1790–1797 (2009)Cite this article

Abstract

Reactive oxygen species (ROS) are continuously produced in the cell as a consequence of aerobic metabolism, and are controlled by several antioxidant mechanisms. An accurate measurement of ROS is essential to evaluate the redox status of the cell, or the effects of molecules with the pro-oxidant or antioxidant activity. Here we report a cytofluorimetric technique for measuring simultaneously, at the single-cell level, hydrogen peroxide and superoxide anion, reduced glutathione (a main intracellular antioxidant) and cell viability. The staining is performed with the fluorescent dyes 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFH-DA), hydroethidine (HE), monobromobimane (MBB) and TO-PRO-3. This analysis is possible with new-generation flow cytometers equipped with several light sources (in our case, four lasers and an UV lamp), which excite different fluorochromes. This approach is extremely useful to study the balance between ROS content and antioxidants in cells receiving different stimuli, and to analyze the relationship between oxidative stress and cell death.

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Figure 1: Separate analysis of the dyes used in this study and modulation of their fluorescence by different agents.
Figure 2: Flow cytometric analysis of ROS and GSH in living cells.
Figure 3: SW872 adipocytes were treated for 14 h with 100 μM stavudine and stained with the four dyes.
Figure 4: CEM cells were treated for 6 or 16 h with 100 μM quercetin and stained with the three indicated dyes.

References

  1. Halliwell, B. Antioxidant defence mechanisms: from the beginning to the end (of the beginning). Free Radic. Res. 31, 261–272 (1999).

    Article  CAS  PubMed  Google Scholar 

  2. Adam-Vizi, V. & Chinopoulos, C. Bioenergetics and the formation of mitochondrial reactive oxygen species. Trends Pharmacol. Sci. 27, 639–645 (2006).

    Article  CAS  PubMed  Google Scholar 

  3. Fridovich, I. Oxygen toxicity: a radical explanation. J. Exp. Biol. 201, 1203–1209 (1998).

    CAS  PubMed  Google Scholar 

  4. Halliwell, B. et al. 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, 231–255 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Petersen, R.B. et al. Signal transduction cascades associated with oxidative stress in Alzheimer's disease. J. Alzheimers Dis. 11, 143–152 (2007).

    Article  CAS  PubMed  Google Scholar 

  6. Multhaup, G. et al. Reactive oxygen species and Alzheimer's disease. Biochem. Pharmacol. 54, 533–539 (1997).

    Article  CAS  PubMed  Google Scholar 

  7. Federico, A. et al. Chronic inflammation and oxidative stress in human carcinogenesis. Int. J. Cancer 121, 2381–2386 (2007).

    Article  CAS  PubMed  Google Scholar 

  8. Ozben, T. Oxidative stress and apoptosis: impact on cancer therapy. J. Pharm. Sci. 96, 2181–2196 (2007).

    Article  CAS  PubMed  Google Scholar 

  9. Shults, C.W. Antioxidants as therapy for Parkinson's disease. Antioxid. Redox Signal. 7, 694–700 (2005).

    Article  CAS  PubMed  Google Scholar 

  10. Nakabeppu, Y. et al. Oxidative damage in nucleic acids and Parkinson's disease. J. Neurosci. Res. 85, 919–934 (2007).

    Article  CAS  PubMed  Google Scholar 

  11. Buxer, S.E. et al. Analytical and numerical techniques for evaluation of free radical damage in cultured cells using imaging cytometry and fluorescent indicators. Methods Enzymol. 300, 256–275 (1999).

    Article  Google Scholar 

  12. Drummen, G.P. et al. C11-BODIPY(581/591), an oxidation-sensitive fluorescent lipid peroxidation probe: (micro)spectroscopic characterization and validation of methodology. Free Radic. Biol. Med. 33, 473–490 (2002).

    Article  CAS  PubMed  Google Scholar 

  13. Brubacher, J.L. et al. Chemically de-acetylated 2′,7′-dichlorodihydrofluorescein diacetate as a probe of respiratory burst activity in mononuclear phagocytes. J. Immunol. Methods 251, 81–91 (2001).

    Article  CAS  PubMed  Google Scholar 

  14. Myhre, O. et al. Evaluation of the probes 2′7′-dichlorofluorescein diacetate, luminol, and lucigenin as indicators of reactive species formation. Biochem. Pharmacol. 65, 1575–1582 (2003).

    Article  CAS  PubMed  Google Scholar 

  15. Hirabayashi, Y. et al. A quantitative assay of oxidative metabolism by neutrophils in whole blood using flow cytometry. J. Immunol. Methods 82, 253–259 (1985).

    Article  CAS  PubMed  Google Scholar 

  16. Rothe, G. et al. Flow cytometric analysis of respiratory burst activity in phagocytes with hydroethidine and 2′,7′-dichlorofluorescein. J. Leukocyte Biol. 47, 440–448 (1990).

    Article  CAS  PubMed  Google Scholar 

  17. Bindokas, V.P. et al. Superoxide production in rat hippocampal neurons: selective imaging with hydroethidine. J. Neurosci. 16, 1324–1326 (1989).

    Article  Google Scholar 

  18. Carter, W.O. et al. Intracellular hydrogen peroxide and superoxide anion detection in endothelial cells. J. Leukoc. Biol. 55, 253–258 (1994).

    Article  CAS  PubMed  Google Scholar 

  19. Pompella, A. et al. The changing faces of glutathione, a cellular protagonist. Biochem. Pharmacol. 66, 1499–1503 (2003).

    Article  CAS  PubMed  Google Scholar 

  20. Hedley, D.W. et al. Evaluation of methods for measuring cellular glutathione content using flow cytometry. Cytometry 15, 349–358 (1994).

    Article  CAS  PubMed  Google Scholar 

  21. Lee-MacAry, A.E. et al. Development of a novel flow cytometric cell-mediated cytotoxicity assay using the fluorophores PKH-26 and TO-PRO-3 iodide. J. Immunol. Methods 252, 83–92 (2001).

    Article  CAS  PubMed  Google Scholar 

  22. Schmid, I. et al. Simultaneous flow cytometric measurement of viability and lymphocyte subset proliferation. J. Immunol. Methods 247, 175–186 (2001).

    Article  CAS  PubMed  Google Scholar 

  23. Troiano, L. et al. Multiparametric analysis of cells with different mitochondrial membrane potential during apoptosis by polychromatic flow cytometry. Nat. Protoc. 2, 2719–2727 (2007).

    Article  CAS  PubMed  Google Scholar 

  24. Lugli, E. et al. Characterization of cells with different mitochondrial membrane potential during apoptosis. Cytometry A 68, 28–35 (2005).

    Article  PubMed  Google Scholar 

  25. Ferraresi, R. et al. Essential requirement of reduced glutathione (GSH) for the anti-oxidant effect of the flavonoid quercetin. Free Radic. Res. 39, 1249–1258 (2005).

    Article  CAS  PubMed  Google Scholar 

  26. Ferraresi, R. et al. Resistance of mtDNA-depleted cells to apoptosis. Cytometry A 73A, 528–537 (2008).

    Article  CAS  Google Scholar 

  27. Misaka, K. et al. Studies on menadione reductase of bakers' yeast. II. Inhibition of the enzyme activity by NAD and stimulation of that by p-chloromercuribenzoate. J. Biochem. 58, 1–6 (1965).

    Article  CAS  PubMed  Google Scholar 

  28. Janiszewski, M. et al. Inhibition of vascular NADH/NADPH oxidase activity by thiol reagents: lack of correlation with cellular glutathione redox status. Free Radic. Biol. Med. 29, 889–899 (2000).

    Article  CAS  PubMed  Google Scholar 

  29. Cossarizza, A. & Moyle, G. Antiretroviral nucleoside and nucleotide analogues and the mitochondria. AIDS 18, 137–151 (2004).

    Article  CAS  PubMed  Google Scholar 

  30. Pinti, M. et al. Hepatoma HepG2 cells as a model for in vitro studies on mitochondrial toxicity of antiviral drugs: which correlation with the patient? J. Biol. Regul. Homeost. Agents 17, 66–71 (2003).

    Google Scholar 

  31. Shiramizu, B. et al. Placenta and cord blood mitochondrial DNA toxicity in HIV-infected women receiving nucleoside reverse transcriptase inhibitors during pregnancy. J. Acquir. Immune Defic. Syndr. 32, 370–374 (2003).

    Article  CAS  PubMed  Google Scholar 

  32. Galluzzi, L. et al. Changes in mitochondrial RNA production in cells treated with nucleoside analogues. Antivir. Ther. 10, 191–195 (2005).

    CAS  PubMed  Google Scholar 

  33. Ferraresi, R. et al. Protective effect of acetyl-L-carnitine against oxidative stress induced by antiretroviral drugs. FEBS Lett. 580, 6612–6616 (2006).

    Article  CAS  PubMed  Google Scholar 

  34. Pinti, M. et al. Anti-HIV drugs and the mitochondria. Biochim. Biophys. Acta 1757, 700–707 (2006).

    Article  CAS  PubMed  Google Scholar 

  35. Rosso, R. et al. Effects of the switch from stavudine to tenovofir in HIV+ children assuming HAART: studies on mitochondrial toxicity and thymic functionality. Pediatrics Infect. Dis. J. 27, 17–21 (2008).

    Article  Google Scholar 

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Acknowledgements

We acknowledge Partec GmbH (Münster, Germany) for continuous technical support. The study is partially supported by the VII Programma Nazionale AIDS, Istituto Superiore di Sanità (Rome, Italy), grant no. 30G.62 to A.C.

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Authors and Affiliations

  1. Department of Biomedical Sciences, University of Modena and Reggio Emilia, Modena, Italy

    Andrea Cossarizza, Roberta Ferraresi, Leonarda Troiano, Erika Roat, Lara Gibellini, Linda Bertoncelli, Milena Nasi & Marcello Pinti

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  1. Andrea Cossarizza
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  8. Marcello Pinti
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Contributions

A.C. designed the study and wrote the paper; R.F., L.B. and M.N. set up the methodologies and performed the experiments; L.T., E.R., L.G. and M.P. set up the methodologies and performed the experiments and wrote the paper.

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Correspondence to Andrea Cossarizza.

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Cossarizza, A., Ferraresi, R., Troiano, L. et al. Simultaneous analysis of reactive oxygen species and reduced glutathione content in living cells by polychromatic flow cytometry. Nat Protoc 4, 1790–1797 (2009). https://doi.org/10.1038/nprot.2009.189

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