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



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.



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.



While analysing ROS-derived fluorescence of commercial hydrocyanine probes, an accurate control of ΔΨm is required.



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.
School of Biochemistry & Cell Biology, University College Cork, Cork, Ireland. Electronic address: a.zhdanov@ucc.ie.
Alexander V
AV
Zhdanov
School of Medicine, Conway Institute, University College Dublin, Dublin, Ireland; University of Aberdeen, Aberdeen, UK.
Gabriella
G
Aviello
School of Medicine, Conway Institute, University College Dublin, Dublin, Ireland.
Ulla G
UG
Knaus
School of Biochemistry & Cell Biology, University College Cork, Cork, Ireland.
Dmitri B
DB
Papkovsky
D000818:Animals; D002232:Carbocyanines / Q000378:metabolism*; D045744:Cell Line, Tumor; D005453:Fluorescence; D045325:HCT116 Cells; D006801:Humans; D053078:Membrane Potential, Mitochondrial / Q000502:physiology*; D051379:Mice; D008810:Mice, Inbred C57BL; D008928:Mitochondria / Q000378:metabolism* / Q000502:physiology*; D009840:Oligomycins / Q000378:metabolism; D010084:Oxidation-Reduction; D017382:Reactive Oxygen Species / Q000378:metabolism*; D013481:Superoxides / Q000378:metabolism
D002232:Carbocyanines; D009840:Oligomycins; D017382:Reactive Oxygen Species; C089187:cyanine dye 3; D013481:Superoxides
Hydro-Cy3; Hydrocyanines; Live cell imaging; Mitochondrial membrane potential; ROS probes