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

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Abstract

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.

Mitochondrial ROS production under cellular stress

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Abbreviations

CPH:

1-hydroxy-3-carboxypyrrolidine

DCF DA:

2′,7′-dichlorodihydrofluorescein diacetate

ESR:

Electron spin resonance

FCS:

Fetal calf serum

NAC:

N-acetylcystein

PPH:

4-phosphonooxy-2,2,6,6-tetramethylpiperidine-N-hydroxyl

ROS:

Reactive oxygen species

t-BHP:

Tert-butyl hydroperoxide

TMRM:

Tetramethylrhodamine methyl ester

TNF-α:

Tumor necrosis factor-alpha

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Acknowledgement

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.

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Authors and Affiliations

  1. Cardiac Surgery Research Laboratory, Department of Heart Surgery, Innsbruck Medical University (IMU), Innrain 66, 6020, Innsbruck, Austria

    Andrey V. Kuznetsov & Michael Grimm

  2. Ludwig Boltzmann Institute for Experimental and Clinical Traumatology, Research Center of AUVA, 1200, Vienna, Austria

    Ingeborg Kehrer, Andrey V. Kozlov & Heinz Redl

  3. Daniel Swarovski Research Laboratory, Department of Visceral, Transplant and Thoracic Surgery, Innsbruck Medical University (IMU), 6020, Innsbruck, Austria

    Martina Haller & Jakob Troppmair

  4. KMT Laboratory, Department of Visceral, Transplant and Thoracic Surgery, Innsbruck Medical University (IMU), 6020, Innsbruck, Austria

    Martin Hermann

Authors
  1. Andrey V. Kuznetsov
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  2. Ingeborg Kehrer
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  3. Andrey V. Kozlov
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  4. Martina Haller
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  5. Heinz Redl
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  6. Martin Hermann
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  7. Michael Grimm
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  8. Jakob Troppmair
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Corresponding author

Correspondence to Andrey V. Kuznetsov.

Additional information

Andrey V. Kuznetsov and Ingeborg Kehrer contributed equally to this study.

The authors are sorry to announce that Ingeborg Kehrer tragically passed away during the preparation of this manuscript.

Published in the special issue Analytical Sciences in Austria with guest editors G. Allmaier W. Buchberger and K. Francesconi.

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Kuznetsov, A.V., Kehrer, I., Kozlov, A.V. et al. Mitochondrial ROS production under cellular stress: comparison of different detection methods. Anal Bioanal Chem 400, 2383–2390 (2011). https://doi.org/10.1007/s00216-011-4764-2

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  • DOI: https://doi.org/10.1007/s00216-011-4764-2

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