Abstract
Hydrogen peroxide (H2O2) produced from mitochondria is intimately involved in human health and disease but is challenging to selectively monitor inside living systems. The fluorescent probe MitoPY1 provides a practical tool for imaging mitochondrial H2O2 and has been demonstrated to function in a variety of diverse cell types. In this chapter, we describe the synthetic preparation of the small-molecule probe MitoPY1, methods for validating this probe in vitro and in live cells, and an example procedure for measuring mitochondrial H2O2 in a cell culture model of Parkinson’s disease.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Desagher S, Martinou JC (2000) Mitochondria as the central control point of apoptosis. Trends Cell Biol 10:369–377
McBride HM, Neuspiel M, Wasiak S (2006) Mitochondria: more than just a powerhouse. Curr Biol 16:R551–R560
Ernstner L, Schatz G (1981) Mitochondria: a historical review. J Cell Biol 91:227s–255s
Petros JA, Baumann AK, Ruiz-Pesini E, Amin MB, Sun CQ, Hall J, Lim SD, Issa MM, Flanders WD, Hosseini SH, Marshall FF, Wallace DC (2005) mtDNA mutations increase tumorigenicity in prostate cancer. Proc Natl Acad Sci U S A 102:719–724
Fato R, Bergamini C, Leoni S, Strocchi P, Lenaz G (2008) Generation of reactive oxygen species by mitochondrial complex I: implications in neurodegeneration. Neurochem Res 33:2487–2501
Lenaz G, Bovina C, D’Aurelio M, Fato R, Formiggini G, Genova ML, Giuliano G, Pich MM, Paolucci U, Castelli GP, Ventura B (2002) Role of mitochondria in oxidative stress and aging. Ann N Y Acad Sci 959:199–213
Veal EA, Day AM, Morgan BA (2007) Hydrogen peroxide sensing and signaling. Mol Cell 26:1–14
Chan J, Dodani SC, Chang CJ (2012) Reaction-based small-molecule fluorescent probes for chemoselective bioimaging. Nat Chem 4:973–984
Lippert AR, Van de Bittner GV, Chang CJ (2011) Boronate oxidation as a bioorthogonal reaction approach for studying the chemistry of hydrogen peroxide in living systems. Acc Chem Res 44:293–804
Chang MCY, Pralle A, Isacoff EY, Chang CJ (2004) A selective, cell-permeable optical probe for hydrogen peroxide in living cells. J Am Chem Soc 126:15392–15393
Miller EW, Albers AE, Pralle A, Isacoff EY, Chang CJ (2005) Boronate-based fluorescent probes for imaging cellular hydrogen peroxide. J Am Chem Soc 127:16652–16659
Dickinson BC, Huynh C, Chang CJ (2010) A palette of fluorescent probes with varying emission colors for imaging hydrogen peroxide signaling in living cells. J Am Chem Soc 132:5906–5915
Murphy MP, Smith RAJ (2007) Targeting antioxidants to mitochondria by conjugation to lipophilic cations. Annu Rev Pharmacol Toxicol 47:629–656
Ross MF, Kelso GF, Blaike FH, James AM, Cochemé HM, Filipovska A, Da Ros T, Hurd TR, Smith RAJ, Murphy MP (2005) Lipophilic triphenylphosphonium cations as tools in mitochondrial bioenergetics and free radical biology. Biochemistry (Mosc) 70:222–230
Dickinson BC, Chang CJ (2008) A targetable fluorescent probe for imaging hydrogen peroxide in the mitochondria of living cells. J Am Chem Soc 130:9638–9639
Dickinson BC, Lin VS, Chang CJ (2013) Preparation and use of MitoPY1 for imaging hydrogen peroxide in live cells. Nat Protoc 8:1249–1259
Chang CJ, Nolan EM, Jaworski J, Okamoto KI, Hayashi Y, Sheng M, Lippard SJ (2004) ZP8, a neuronal zinc sensor with improved dynamic range; imaging zinc in hippocampal slices with two-photon microscopy. Inorg Chem 43:6774–6779
Lin TK, Hughes G, Muratovska A, Blaikie FH, Brookes PS, Darley-Usmar V, Smith RAJ, Murphy MP (2002) Specific modification of mitochondrial protein thiols in response to oxidative stress. J Biol Chem 277:17048–17056
Still WC, Kahn M, Mitra A (1978) Rapid chromatographic techniques for preparative separation with moderate resolution. J Org Chem 43:2923–2925
Pendergrass W, Wolf N, Poot M (2004) Efficacy of MitoTracker Green™ and CMXrosamine to measure changes in mitochondrial membrane potentials in living cells and tissues. Cytometry A 61A:162–169
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2015 Springer Science+Business Media New York
About this protocol
Cite this protocol
Lippert, A.R., Dickinson, B.C., New, E.J. (2015). Imaging Mitochondrial Hydrogen Peroxide in Living Cells. In: Weissig, V., Edeas, M. (eds) Mitochondrial Medicine. Methods in Molecular Biology, vol 1264. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2257-4_21
Download citation
DOI: https://doi.org/10.1007/978-1-4939-2257-4_21
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-2256-7
Online ISBN: 978-1-4939-2257-4
eBook Packages: Springer Protocols