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Simultaneous detection of apoptosis and mitochondrial superoxide production in live cells by flow cytometry and confocal microscopy

Nature Protocols volume 2, pages 2295–2301 (2007)Cite this article

Abstract

Annexin V and Sytox Green are widely used markers to evaluate apoptosis in various cell types using flow cytometry and fluorescent microscopy. Recently, a novel fluoroprobe MitoSOX Red was introduced for selective detection of superoxide in the mitochondria of live cells and was validated for confocal microscopy and flow cytometry. This protocol describes simultaneous measurements of mitochondrial superoxide generation with apoptotic markers (Annexin V and Sytox Green) by both flow cytometry and confocal microscopy in endothelial cell lines. The advantages of the described flow cytometry method over other cell-based techniques are the tremendous speed (1–2 h), exquisite precision and the possibility of simultaneous quantitative measurements of mitochondrial superoxide generation and apoptotic (and other) markers, with maximal preservation of cellular functions. This method combined with fluorescent microscopy may be very useful to reveal important spatial–temporal changes in mitochondrial superoxide production and execution of programmed cell death in virtually any cell type.

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Figure 1: Simultaneous determination of apoptotic markers and mitochondrial superoxide formation by flow cytometry and confocal microscopy following DOX treatment.
Figure 2: Determination of mitochondrial superoxide production measured by flow cytometry and confocal microscopy using MitoSOX.

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References

  1. Fridovich, I. Superoxide radical and superoxide dismutases. Annu. Rev. Biochem. 64, 97–112 (1995).

    Article  CAS  PubMed  Google Scholar 

  2. Griendling, K.K., Sorescu, D. & Ushio-Fukai, M. NAD(P)H oxidase: role in cardiovascular biology and disease. Circ. Res. 86, 494–501 (2000).

    Article  CAS  PubMed  Google Scholar 

  3. Csiszar, A., Pacher, P., Kaley, G. & Ungvari, Z. Role of oxidative and nitrosative stress, longevity genes and poly(ADP-ribose) polymerase in cardiovascular dysfunction associated with aging. Curr. Vasc. Pharmacol. 3, 285–291 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Pacher, P., Nivorozhkin, A. & Szabo, C. Therapeutic effects of xanthine oxidase inhibitors: renaissance half a century after the discovery of allopurinol. Pharmacol. Rev. 58, 87–114 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Pacher, P., Beckman, J.S. & Liaudet, L. Nitric oxide and peroxynitrite in health and disease. Physiol. Rev. 87, 315–424 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Gomes, A., Fernandes, E. & Lima, J.L. Fluorescence probes used for detection of reactive oxygen species. J. Biochem. Biophys. Methods 65, 45–80 (2005).

    Article  CAS  PubMed  Google Scholar 

  7. Robinson, K.M. et al. Selective fluorescent imaging of superoxide in vivo using ethidium-based probes. Proc. Natl. Acad. Sci. USA 103, 15038–15043 (2006).

    Article  CAS  PubMed  Google Scholar 

  8. Soh, N. Recent advances in fluorescent probes for the detection of reactive oxygen species. Anal. Bioanal. Chem. 386, 532–543 (2006).

    Article  CAS  PubMed  Google Scholar 

  9. Zhao, H. et al. Superoxide reacts with hydroethidine but forms a fluorescent product that is distinctly different from ethidium: potential implications in intracellular fluorescence detection of superoxide. Free Radic. Biol. Med. 34, 1359–1368 (2003).

    Article  CAS  PubMed  Google Scholar 

  10. Zhao, H. et al. Detection and characterization of the product of hydroethidine and intracellular superoxide by HPLC and limitations of fluorescence. Proc. Natl. Acad. Sci. USA 102, 5727–5732 (2005).

    Article  CAS  PubMed  Google Scholar 

  11. Rezvani, H.R. et al. Hypoxia-inducible factor-1alpha, a key factor in the keratinocyte response to UVB exposure. J. Biol. Chem. 282, 16413–16422 (2007).

    Article  CAS  PubMed  Google Scholar 

  12. Pehar, M. et al. Mitochondrial superoxide production and nuclear factor erythroid 2-related factor 2 activation in p75 neurotrophin receptor-induced motor neuron apoptosis. J. Neurosci. 27, 7777–7785 (2007).

    Article  CAS  PubMed  Google Scholar 

  13. Johnson-Cadwell, L.I., Jekabsons, M.B., Wang, A., Polster, B.M. & Nicholls, D.G. 'Mild uncoupling' does not decrease mitochondrial superoxide levels in cultured cerebellar granule neurons but decreases spare respiratory capacity and increases toxicity to glutamate and oxidative stress. J. Neurochem. 101, 1619–1631 (2007).

    Article  CAS  PubMed  Google Scholar 

  14. Abramov, A.Y., Scorziello, A. & Duchen, M.R. Three distinct mechanisms generate oxygen free radicals in neurons and contribute to cell death during anoxia and reoxygenation. J. Neurosci. 27, 1129–1138 (2007).

    Article  CAS  PubMed  Google Scholar 

  15. Zimmerman, M.C., Oberley, L.W. & Flanagan, S.W. Mutant SOD1-induced neuronal toxicity is mediated by increased mitochondrial superoxide levels. J. Neurochem. (2007).

  16. Fauconnier, J. et al. Effects of palmitate on Ca2+ handling in adult control and ob/ob cardiomyocytes: impact of mitochondrial reactive oxygen species. Diabetes 56, 1136–1142 (2007).

    Article  CAS  PubMed  Google Scholar 

  17. Iuso, A. et al. Dysfunctions of cellular oxidative metabolism in patients with mutations in the NDUFS1 and NDUFS4 genes of complex I. J. Biol. Chem. 281, 10374–10380 (2006).

    Article  CAS  PubMed  Google Scholar 

  18. Hawkins, B.J., Madesh, M., Kirkpatrick, C.J. & Fisher, A.B. Superoxide flux in endothelial cells via the chloride channel-3 mediates intracellular signaling. Mol. Biol. Cell 18, 2002–2012 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Mukhopadhyay, P., Rajesh, M., Yoshihiro, K., Hasko, G. & Pacher, P. Simple quantitative detection of mitochondrial superoxide production in live cells. Biochem. Biophys. Res. Commun. 358, 203–208 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Brookes, P.S. et al. The triterpenoid 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid and its derivatives elicit human lymphoid cell apoptosis through a novel pathway involving the unregulated mitochondrial permeability transition pore. Cancer Res. 67, 1793–1802 (2007).

    Article  CAS  PubMed  Google Scholar 

  21. Zhang, Y., Soboloff, J., Zhu, Z. & Berger, S.A. Inhibition of Ca2+ influx is required for mitochondrial reactive oxygen species-induced endoplasmic reticulum Ca2+ depletion and cell death in leukemia cells. Mol. Pharmacol. 70, 1424–1434 (2006).

    Article  CAS  PubMed  Google Scholar 

  22. Lund, K.C., Peterson, L.L. & Wallace, K.B. The absence of a universal mechanism of mitochondrial toxicity by nucleoside analogs. Antimicrob. Agents Chemother. (2007).

  23. Payne, C.M. et al. Deoxycholate induces mitochondrial oxidative stress and activates NF-{kappa}B through multiple mechanisms in HCT-116 colon epithelial cells. Carcinogenesis 28, 215–222 (2007).

    Article  CAS  PubMed  Google Scholar 

  24. Dvorak, K. et al. Bile acids in combination with low pH induce oxidative stress and oxidative DNA damage: relevance to the pathogenesis of Barrett's oesophagus. Gut 56, 763–771 (2007).

    Article  CAS  PubMed  Google Scholar 

  25. Rajesh, M. et al. Cannabidiol attenuates high glucose-induced endothelial cell inflammatory response and barrier disruption. Am. J. Physiol. Heart Circ. Physiol. 293, H610–H619 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Ungvari, Z. et al. Increased mitochondrial H2O2 production promotes endothelial NF-κB activation in aged rat arteries. Am. J. Physiol. Heart Circ. Physiol. 293, H37–H47 (2007).

    Article  CAS  PubMed  Google Scholar 

  27. Poot, M., Gibson, L.L. & Singer, V.L. Detection of apoptosis in live cells by MitoTracker red CMXRos and SYTO dye flow cytometry. Cytometry 27, 358–364 (1997).

    Article  CAS  PubMed  Google Scholar 

  28. van Genderen, H. et al. In vitro measurement of cell death with the annexin A5 affinity assay. Nat. Protoc. 1, 363–367 (2006).

    Article  CAS  PubMed  Google Scholar 

  29. Mukhopadhyay, P. et al. Pharmacological inhibition of CB1 cannabinoid receptor protects against doxorubicin-induced cardiotoxicity. J. Am. Coll. Cardiol. 50, 528–536 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

This research was supported by the Intramural Research Program of NIH/NIAAA (to P.P.). M.M. was supported by AHA (0530087N) and NCRR (ISIORR022511-01A1) grants. B.J.H. was supported by a fellowship from the Hypertension Association. We are indebted to Professors Joseph S. Beckman and Balaraman Kalyanaraman for reading the protocol and for valuable comments.

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Authors and Affiliations

  1. Section on Oxidative Stress Tissue Injury, Laboratories of Physiological Studies, National Institutes of Health/NIAAA, Bethesda, 20892, Maryland, USA

    Partha Mukhopadhyay, Mohanraj Rajesh & Pál Pacher

  2. Department of Surgery, UMDNJ-New Jersey Medical School, Newark, 07103, New Jersey, USA

    György Haskó

  3. Department of Cancer Biology, Institute for Environmental Medicine, University of Pennsylvania, Philadelphia, 19104, Pennsylvania, USA

    Brian J Hawkins & Muniswamy Madesh

Authors
  1. Partha Mukhopadhyay
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Correspondence to Pál Pacher.

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Mukhopadhyay, P., Rajesh, M., Haskó, G. et al. Simultaneous detection of apoptosis and mitochondrial superoxide production in live cells by flow cytometry and confocal microscopy. Nat Protoc 2, 2295–2301 (2007). https://doi.org/10.1038/nprot.2007.327

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