What does the commonly used DCF test for oxidative stress really show?

@article{Karlsson2010WhatDT,
  title={What does the commonly used DCF test for oxidative stress really show?},
  author={Markus Karlsson and Tino Kurz and Ulf T. Brunk and Sven Erik G. Nilsson and Christina I Frennesson},
  journal={The Biochemical journal},
  year={2010},
  volume={428 2},
  pages={
          183-90
        }
}
H(2)DCF-DA (dihydrodichlorofluorescein diacetate) is widely used to evaluate 'cellular oxidative stress'. After passing through the plasma membrane, this lipophilic and non-fluorescent compound is de-esterified to a hydrophilic alcohol [H(2)DCF (dihydrodichlorofluorescein)] that may be oxidized to fluorescent DCF (2',7'-dichlorofluorescein) by a process usually considered to involve ROS (reactive oxygen species). It is, however, not always recognized that, being a hydrophilic molecule, H(2)DCF… 

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References

SHOWING 1-10 OF 38 REFERENCES

Cytochrome C is a potent catalyst of dichlorofluorescin oxidation: implications for the role of reactive oxygen species in apoptosis.

TLDR
It is concluded that the DCF fluorescence observed in dying cells is a reflection of increased cytosolic cytochrome c, and suggested that the suppression of DCF formation by the anti-apoptotic oncoprotein Bcl-2, which has been suggested to have antioxidant properties, can be explained on the basis of its prevention of mitochondrial cy tochrome c release.

Apoptosis induced by exposure to a low steady-state concentration of H2O2 is a consequence of lysosomal rupture.

TLDR
The results reinforce earlier findings and strongly suggest that lysosomal rupture is an early upstream initiating event, and a consequence of intralysosome iron-catalysed oxidative processes, when apoptosis is induced by oxidative stress.

Localization of dichlorofluorescin in cardiac myocytes: implications for assessment of oxidative stress.

  • L. SwiftN. Sarvazyan
  • Biology, Chemistry
    American journal of physiology. Heart and circulatory physiology
  • 2000
TLDR
The study provides an information base essential for the interpretation and design of experiments with DCF as a marker of oxidative stress in cardiac muscle and reveals preferential localization of the probe in mitochondria.

Measuring reactive species and oxidative damage in vivo and in cell culture: how should you do it and what do the results mean?

TLDR
Methods used to trap RS, including spin trapping and aromatic hydroxylation, are critically examined, with a particular emphasis on those methods applicable to human studies and methods used to detect RS in cell culture.

Does the calcein-AM method assay the total cellular 'labile iron pool' or only a fraction of it?

TLDR
It is shown that the calcein-AM method does not capture lysosomal low-mass iron and, therefore, that the method seriously underestimates total cellular labile iron.

Flow cytometric studies of oxidative product formation by neutrophils: a graded response to membrane stimulation.

TLDR
The data indicate that the DCFH oxidation assay is quantitatively related to the oxidative metabolic burst of PMNL, and they strongly suggest that the reaction is mediated by H2O2 generated by the PMNL.

Intralysosomal iron chelation protects against oxidative stress‐induced cellular damage

TLDR
The results lend strength to the concept that lysosomal membranes, normally exposed to redox‐active iron in high concentrations, are initial targets of oxidant damage and support the idea that chelators selectively targeted to the lysOSomal compartment may have therapeutic utility in diminishing oxidant‐mediated cell injury.