Cellular ROS imaging with hydro-Cy3 dye is strongly influenced by mitochondrial membrane potential

dc.contributor.authorZhdanov, Alexander V.
dc.contributor.authorAviello, Gabriella
dc.contributor.authorKnaus, Ulla G.
dc.contributor.authorPapkovsky, Dmitri B.
dc.contributor.funderScience Foundation Irelanden
dc.date.accessioned2016-12-09T14:40:27Z
dc.date.available2016-12-09T14:40:27Z
dc.date.issued2016-11-04
dc.date.updated2016-12-09T14:31:09Z
dc.description.abstractBackground: Hydrocyanines are widely used as fluorogenic probes to monitor reactive oxygen species (ROS) generation in cells. Their brightness, stability to autoxidation and photobleaching, large signal change upon oxidation, pH independence and red/near infrared emission are particularly attractive for imaging ROS in live tissue. Methods: Using confocal fluorescence microscopy we have examined an interference of mitochondrial membrane potential (ΔΨm) with fluorescence intensity and localisation of a commercial hydro-Cy3 probe in respiring and non-respiring colon carcinoma HCT116 cells. Results: We found that the oxidised (fluorescent) form of hydro-Cy3 is highly homologous to the common ΔΨm-sensitive probe JC-1, which accumulates and aggregates only in ‘energised’ negatively charged mitochondrial matrix. Therefore, hydro-Cy3 oxidised by hydroxyl and superoxide radicals tends to accumulate in mitochondrial matrix, but dissipates and loses brightness as soon as ΔΨm is compromised. Experiments with mitochondrial inhibitor oligomycin and uncoupler FCCP, as well as a common ROS producer paraquat demonstrated that signals of the oxidised hydro-Cy3 probe rapidly and strongly decrease upon mitochondrial depolarisation, regardless of the rate of cellular ROS production. Conclusions: While analysing ROS-derived fluorescence of commercial hydrocyanine probes, an accurate control of ΔΨm is required. General significance: If not accounted for, non-specific effect of mitochondrial polarisation state on the behaviour of oxidised hydrocyanines can cause artefacts and data misinterpretation in ROS studies.en
dc.description.sponsorshipScience Foundation Ireland (SFI grant 12/RC/2276)en
dc.description.statusPeer revieweden
dc.description.versionAccepted Versionen
dc.format.mimetypeapplication/pdfen
dc.identifier.citationZhdanov, A. V., Aviello, G., Knaus, U. G. and Papkovsky, D. B. (2017) 'Cellular ROS imaging with hydro-Cy3 dye is strongly influenced by mitochondrial membrane potential', Biochimica et Biophysica Acta (BBA) - General Subjects, 1861(2), pp. 198-204. doi:10.1016/j.bbagen.2016.10.023en
dc.identifier.doi10.1016/j.bbagen.2016.10.023
dc.identifier.endpage204en
dc.identifier.issn0304-4165
dc.identifier.issued22en
dc.identifier.journaltitleBiochimica et Biophysica Acta (BBA) - General Subjectsen
dc.identifier.startpage198en
dc.identifier.urihttps://hdl.handle.net/10468/3368
dc.identifier.volume1861en
dc.language.isoenen
dc.publisherElsevieren
dc.rights© 2016, Elsevier. This manuscript version is made available under the CC-BY-NC-ND 4.0 license http://creativecommons.org/licenses/by-nc-nd/4.0/en
dc.rights.urihttp://creativecommons.org/licenses/by-nc-nd/4.0/en
dc.subjectROS probesen
dc.subjectHydrocyaninesen
dc.subjectHydro-Cy3en
dc.subjectMitochondrial membrane potentialen
dc.subjectLive cell imagingen
dc.titleCellular ROS imaging with hydro-Cy3 dye is strongly influenced by mitochondrial membrane potentialen
dc.typeArticle (peer-reviewed)en
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