A Ruthenium Probe for Cell Viability Measurement Using Flow Cytometry, Confocal Microscopy and Time-resolved Luminescence¶

Abstract

The capability of the new luminescent probe (dibenzo[h,j] dipyrido[3,2-a:2',3'-c]phenazine)bis(2,2'-bipyridine)ruthenium(II) dication, (RB2Z), to discriminate live and dead cells has been tested on rat hepatocytes and mouse lymphocytes. RB2Z-stained cells were analyzed using flow cytometry, fluorescence (confocal) microscopy and time-resolved luminescence measurements. The established viability probes propidium iodide (PI) and SYTOX green (SG) were used as controls. The intense luminescence of RB2Z at 606 nm is localized in the nucleus of nonviable cells. Viability measurements by flow cytometry and fluorescence microscopy using RB2Z as dead-cell marker yield the same results as PI and SG. The luminescence lifetime of RB2Z also displays sensitivity to cell viability (0.45 and 0.82 microsecond in presence of fully viable and dead cells, respectively). This ruthenium complex is photostable under laser sources and its 200 nm Stokes shift facilitates multicolor labeling experiments in flow cytometry and fluorescence microscopy. Unlike the currently available probes, the long-lived excited state of RB2Z also allows assays based on luminescence decay measurements.

Lipophilic ruthenium complexes with tuned cell membrane affinity and photoactivated uptake

“…Barton et al have recently shown the usefulness of this effect for studying the cellular uptake and nuclear localization of Ru(II) dppz complexes [3,4]. Similar complexes have also been used as probes for cell viability and nuclear staining [5,6]. We have previously shown that Ru(II) dppz complexes, made more hydrophobic by substitution with alkyl ether chains, are versatile as photophysical probes for phospholipid bilayers [7,8].…”

Therapeutic Potential of Photochemically Active Metal Complexes based on Interaction with Enzymes

“…Luminescence using viable cells was only detectable after addition of Triton X-100 as surfactant. It may therefore appear that 6 can not migrate across an intact fully functional cellular membrane, but is capable of binding to the nuclear DNA in dead cells or cells with damaged membranes [34]. The related dinuclear ruthenium 7 posesses an overall charge of 4+ and shows very high binding affinities to DNA, Fig.…”

Quantitative Investigation of Biomolecular Interactions in Crowded Media by Fluorescence Spectroscopy, a Good Choice

“…The global tumbling of dimeric apoMb was accessible using ANS as probe (lifetime 15ns) but not by fluorescein, due to its short lifetime ( 3ns), and its local wobbling. The development of ruthenium derivatives [89,90] displaying longer lifetimes (from 400 to 700 ns) may contribute to improve the performance of fluorescence polarization experiments in crowding conditions. [56]), which appears as a residual anisotropy in the apoMb-Fl decays.…”