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Preparation and use of MitoPY1 for imaging hydrogen peroxide in mitochondria of live cells

Nature Protocols volume 8, pages 1249–1259 (2013)Cite this article

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Abstract

Mitochondria peroxy yellow 1 (MitoPY1) is a small-molecule fluorescent probe that selectively tracks to the mitochondria of live biological specimens and responds to local fluxes of hydrogen peroxide (H2O2) by a turn-on fluorescence enhancement. This bifunctional dye uses a triphenylphosphonium targeting group and a boronate-based molecular switch to selectively respond to H2O2 over competing reactive oxygen species (ROS) within the mitochondria. MitoPY1 can be used to measure mitochondrial H2O2 levels in both cell culture and tissue models. In this protocol, we describe the synthesis of MitoPY1 and how to use this chemical tool to visualize mitochondrial H2O2 in live cells. The preparation of MitoPY1 is anticipated to take 7–10 d, and assays involving microscopy of cultured mammalian cells can be performed in 1–2 d.

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Figure 1: Mitochondria Peroxy Yellow 1 (MitoPY1).
Figure 2: Fluorescence turn-on response of 5 μM MitoPY1 to H2O2.
Figure 3: Imaging mitochondrial H2O2 in live cells with MitoPY1.

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Acknowledgements

This work was supported by the US National Institutes of Health (NIH) (GM 79465). C.J.C. is an Investigator with the Howard Hughes Medical Institute. B.C.D. is a Fellow of the Jane Coffin Childs Memorial Fund for Medical Research. V.S.L. acknowledges Amgen for a graduate fellowship.

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Authors and Affiliations

  1. Department of Chemistry and Molecular and Cell Biology, Howard Hughes Medical Institute, University of California, Berkeley, California, USA

    Bryan C Dickinson, Vivian S Lin & Christopher J Chang

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  1. Bryan C Dickinson
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  2. Vivian S Lin
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  3. Christopher J Chang
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Contributions

B.C.D. and V.S.L. performed experiments. B.C.D., V.S.L. and C.J.C. designed experiments and wrote the paper.

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Correspondence to Christopher J Chang.

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The authors declare no competing financial interests.

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Dickinson, B., Lin, V. & Chang, C. Preparation and use of MitoPY1 for imaging hydrogen peroxide in mitochondria of live cells. Nat Protoc 8, 1249–1259 (2013). https://doi.org/10.1038/nprot.2013.064

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