Analytical and Bioanalytical Chemistry

, Volume 400, Issue 8, pp 2383–2390 | Cite as

Mitochondrial ROS production under cellular stress: comparison of different detection methods

  • Andrey V. Kuznetsov
  • Ingeborg Kehrer
  • Andrey V. Kozlov
  • Martina Haller
  • Heinz Redl
  • Martin Hermann
  • Michael Grimm
  • Jakob Troppmair
Original Paper


Reactive oxygen species (ROS) are involved in the regulation of many physiological processes. However, overproduction of ROS under various cellular stresses results in cell death and organ injury and thus contributes to a broad spectrum of diseases and pathological conditions. The existence of different cellular sources for ROS and the distinct properties of individual ROS (their reactivity, lifetime, etc.) require adequate detection methods. We therefore compared different models of cellular stress and various ROS-sensitive dyes—2′,7′-dichlorodihydrofluorescein diacetate (DCF-DA), MitoSOX™, and MitoTracker® red CM-H2XRos—using a confocal fluorescent imaging approach, which has the advantage of not only detecting but also of localizing intracellular sources for ROS. Confocal acquisition of DCF-DA fluorescence can be combined with ROS detection by the mitochondria-specific probes MitoSOX™ and MitoTracker® red CM-H2XRos. Specificity was controlled using various antioxidants such as Trolox and N-acetylcysteine. Using different fluorescent ROS-sensitive probes, we detected higher ROS production equally under cell starvation (IL-3 or serum depletion), hypoxia–reoxygenation, or treatment of cells with prooxidants. The detected increase in ROS was approximately threefold in IL-3-depleted 32D cells, approximately 3.5-fold in serum-deprived NIH cells, and 2.5-fold to threefold in hypoxic HL-1 cells, and these findings agree well with previously published spectrofluorometric measurements. In some cases, electron spin resonance (ESR) spectroscopy was used for the validation of results from confocal fluorescent imaging. Our data show that confocal fluorescent imaging and ESR data are in good agreement. Under cellular stress, mitochondrial ROS are released into the cytoplasm and may participate in many processes, but they do not escape from the cell.

Online abstract

Mitochondrial ROS production under cellular stress


Confocal fluorescent imaging Cell stress Electron spin resonance Laser scanning microscopy Mitochondria Reactive oxygen species 





2′,7′-dichlorodihydrofluorescein diacetate


Electron spin resonance


Fetal calf serum






Reactive oxygen species


Tert-butyl hydroperoxide


Tetramethylrhodamine methyl ester


Tumor necrosis factor-alpha



This work was supported by a research grant from the Austrian Science Fund (FWF): [P 22080-B20] to A.V.K., and by funds from the Jubiläumsfond der Österreichisschen Nationalbank (OeNB) to J.T.


  1. 1.
    Droge W (2002) Physiol Rev 82:47–95Google Scholar
  2. 2.
    Hermann M, Kuznetsov A, Maglione M, Smigelskaite J, Margreiter R, Troppmair J (2008) Cell Commun Signal 6:4CrossRefGoogle Scholar
  3. 3.
    Kamata H, Honda S, Maeda S, Chang L, Hirata H, Karin M (2005) Cell 120:649–661CrossRefGoogle Scholar
  4. 4.
    Kowaltowski AJ, de Souza-Pinto NC, Castilho RF, Vercesi AE (2009) Free Radic Biol Med 47:333–343CrossRefGoogle Scholar
  5. 5.
    Magder S (2006) Crit Care 10:208CrossRefGoogle Scholar
  6. 6.
    Thannickal VJ, Fanburg BL (2000) Am J Physiol Lung Cell Mol Physiol 279:L1005–L1028Google Scholar
  7. 7.
    Zhang DX, Gutterman DD (2007) Am J Physiol Heart Circ Physiol 292:H2023–H2031CrossRefGoogle Scholar
  8. 8.
    Akao M, O'Rourke B, Teshima Y, Seharaseyon J, Marban E (2003) Circ Res 92:186–194CrossRefGoogle Scholar
  9. 9.
    Balaban RS, Nemoto S, Finkel T (2005) Cell 120:483–495CrossRefGoogle Scholar
  10. 10.
    Chandel NS, Maltepe E, Goldwasser E, Mathieu CE, Simon MC, Schumacker PT (1998) Proc Natl Acad Sci USA 95:11715–11720CrossRefGoogle Scholar
  11. 11.
    Cruthirds DL, Novak L, Akhi KM, Sanders PW, Thompson JA, Millan-Crow LA (2003) Arch Biochem Biophys 412:27–33CrossRefGoogle Scholar
  12. 12.
    Halliwell B (1993) Haemostasis 23(Suppl 1):118–126Google Scholar
  13. 13.
    Rodriguez R, Redman R (2005) Proc Natl Acad Sci USA 102:3175–3176CrossRefGoogle Scholar
  14. 14.
    Semenza GL (2000) Circ Res 86:117–118Google Scholar
  15. 15.
    Thannickal VJ (2003) Am J Physiol Lung Cell Mol Physiol 284:L24–L25Google Scholar
  16. 16.
    Benhar M, Engelberg D, Levitzki A (2002) EMBO Rep 3:420–425CrossRefGoogle Scholar
  17. 17.
    Gianni D, Taulet N, Dermardirossian C, Bokoch GM (2010) Mol Biol Cell 21:4287–4298Google Scholar
  18. 18.
    Orrenius S, Gogvadze V, Zhivotovsky B (2007) Annu Rev Pharmacol Toxicol 47:143–183CrossRefGoogle Scholar
  19. 19.
    Kuznetsov AV, Smigelskaite J, Doblander C, Janakiraman M, Hermann M, Wurm M, Scheidl SF, Sucher R, Deutschmann A, Troppmair J (2008) Mol Cell Biol 28:2304–2313CrossRefGoogle Scholar
  20. 20.
    Bindokas VP, Kuznetsov A, Sreenan S, Polonsky KS, Roe MW, Philipson LH (2003) J Biol Chem 278:9796–9801CrossRefGoogle Scholar
  21. 21.
    Diaz G, Liu S, Isola R, Diana A, Falchi AM (2003) Histochem Cell Biol 120:319–325CrossRefGoogle Scholar
  22. 22.
    Mukhopadhyay P, Rajesh M, Hasko G, Hawkins BJ, Madesh M, Pacher P (2007) Nat Protoc 2:2295–2301CrossRefGoogle Scholar
  23. 23.
    Wang W, Fang H, Groom L, Cheng A, Zhang W, Liu J, Wang X, Li K, Han P, Zheng M, Yin J, Wang W, Mattson MP, Kao JP, Lakatta EG, Sheu SS, Ouyang K, Chen J, Dirksen RT, Cheng H (2008) Cell 134:279–290CrossRefGoogle Scholar
  24. 24.
    Zorov DB, Filburn CR, Klotz LO, Zweier JL, Sollott SJ (2000) J Exp Med 192:1001–1014CrossRefGoogle Scholar
  25. 25.
    Mariappan N, Elks CM, Fink B, Francis J (2009) Free Radic Biol Med 46:462–470CrossRefGoogle Scholar
  26. 26.
    O'Malley Y, Fink BD, Ross NC, Prisinzano TE, Sivitz WI (2006) J Biol Chem 281:39766–39775CrossRefGoogle Scholar
  27. 27.
    Buettner GR (1987) Free Radic Biol Med 3:259–303CrossRefGoogle Scholar
  28. 28.
    Dikalov S, Griendling KK, Harrison DG (2007) Hypertension 49:717–727CrossRefGoogle Scholar
  29. 29.
    Kozlov AV, Szalay L, Umar F, Fink B, Kropik K, Nohl H, Redl H, Bahrami S (2003) Free Radic Biol Med 34:1555–1562CrossRefGoogle Scholar
  30. 30.
    Takeshita K, Ozawa T (2004) J Radiat Res Tokyo 45:373–384CrossRefGoogle Scholar
  31. 31.
    Brookes PS, Yoon Y, Robotham JL, Anders MW, Sheu SS (2004) Am J Physiol Cell Physiol 287:C817–C833CrossRefGoogle Scholar
  32. 32.
    Han D, Williams E, Cadenas E (2001) Biochem J 353:411–416CrossRefGoogle Scholar
  33. 33.
    Murphy MP (2009) Biochem J 417:1–13CrossRefGoogle Scholar
  34. 34.
    Turrens JF (2003) J Physiol 552:335–344CrossRefGoogle Scholar
  35. 35.
    Chandel NS, McClintock DS, Feliciano CE, Wood TM, Melendez JA, Rodriguez AM, Schumacker PT (2000) J Biol Chem 275:25130–25138CrossRefGoogle Scholar
  36. 36.
    Guzy RD, Hoyos B, Robin E, Chen H, Liu L, Mansfield KD, Simon MC, Hammerling U, Schumacker PT (2005) Cell Metab 1:401–408CrossRefGoogle Scholar
  37. 37.
    Kudin AP, Bimpong-Buta NY, Vielhaber S, Elger CE, Kunz WS (2004) J Biol Chem 279:4127–4135CrossRefGoogle Scholar
  38. 38.
    Lambert AJ, Brand MD (2004) Biochem J 382:511–517CrossRefGoogle Scholar
  39. 39.
    Prata C, Maraldi T, Fiorentini D, Zambonin L, Hakim G, Landi L (2008) Free Radic Res 42:405–414CrossRefGoogle Scholar
  40. 40.
    Geiszt M, Leto TL (2004) J Biol Chem 279:51715–51718CrossRefGoogle Scholar
  41. 41.
    Guzy RD, Schumacker PT (2006) Exp Physiol 91:807–819CrossRefGoogle Scholar
  42. 42.
    Rieske JS, Lipton SH, Baum H, Silman HI (1967) J Biol Chem 242:4888–4896Google Scholar
  43. 43.
    Gupta S (2002) J Clin Immunol 22:185–194CrossRefGoogle Scholar
  44. 44.
    Kozlov AV, Staniek K, Haindl S, Piskernik C, Ohlinger W, Gille L, Nohl H, Bahrami S, Redl H (2006) Am J Physiol Gastrointest Liver Physiol 290:G543–G549CrossRefGoogle Scholar
  45. 45.
    Sucher R, Gehwolf P, Kaier T, Hermann M, Maglione M, Oberhuber R, Ratschiller T, Kuznetsov AV, Bosch F, Kozlov AV, Ashraf MI, Schneeberger S, Brandacher G, Ollinger R, Margreiter R, Troppmair J (2009) Transpl Int 22:922–930CrossRefGoogle Scholar
  46. 46.
    Schumacker PT (2003) Adv Exp Med Biol 543:57–71Google Scholar
  47. 47.
    Kozlov AV, Gille L, Miller I, Piskernik C, Haindl S, Staniek K, Nohl H, Bahrami S, Ohlinger W, Gemeiner M, Redl H (2007) Biochem Biophys Res Commun 352:91–96CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2011

Authors and Affiliations

  • Andrey V. Kuznetsov
    • 1
  • Ingeborg Kehrer
    • 2
  • Andrey V. Kozlov
    • 2
  • Martina Haller
    • 3
  • Heinz Redl
    • 2
  • Martin Hermann
    • 4
  • Michael Grimm
    • 1
  • Jakob Troppmair
    • 3
  1. 1.Cardiac Surgery Research Laboratory, Department of Heart SurgeryInnsbruck Medical University (IMU)InnsbruckAustria
  2. 2.Ludwig Boltzmann Institute for Experimental and Clinical Traumatology, Research Center of AUVAViennaAustria
  3. 3.Daniel Swarovski Research Laboratory, Department of Visceral, Transplant and Thoracic SurgeryInnsbruck Medical University (IMU)InnsbruckAustria
  4. 4.KMT Laboratory, Department of Visceral, Transplant and Thoracic SurgeryInnsbruck Medical University (IMU)InnsbruckAustria

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