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

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.

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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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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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