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

Alexander V Zhdanov, Gabriella Aviello, Ulla G Knaus, Dmitri B Papkovsky

Research output: Contribution to journalArticlepeer-review

13 Citations (Scopus)


BACKGROUND: 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.

Original languageEnglish
Pages (from-to)198-204
Number of pages7
JournalBiochimica et Biophysica Acta
Issue number2
Early online date4 Nov 2016
Publication statusPublished - Feb 2017

Bibliographical note

Authors thank Professor P.M. Hwang (NIH) for kindly providing with HCT116 SCO2-/- cells. Financial support by Science Foundation Ireland grant 12/RC/2276 is gratefully acknowledged.


  • ROS probes
  • hydrocyanines
  • Hydro-Cy3
  • mitochondrial membrane potential
  • live cell imaging


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