A ruthenium probe for cell viability measurement using flow cytometry, confocal microscopy and time-resolved luminescence

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 dis criminate live and dead cells has been tested on rat hepatocytes and mouse lymphocytes. RB2Z-stained cells were analyzed using dow cytometry, fluoresc ence (confocal) microscopy and time-resolved luminescence measurements. The established viability probes propidium iodide (PI) and SYTOX(R) green (SG) were used as controls. The intense luminescence of RB2Z at 606 nm is local ized in the nucleus of nonviable cells. Viability measurements by flow cyto metry 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 mu s in presence of fully viab le and dead cells, respectively). This ruthenium complex is photostable und er laser sources and its 200 nm Stokes shift facilitates multicolor labelin g experiments in flow cytometry and fluorescence microscopy, Unlike the cur rently available probes, the long-lived excited state of RB2Z also allows a ssays based on luminescence decay measurements.