28 q 2000 American Society for Photobiology 0031-8655/00 $5.00 1 0.00 Photochemistry and Photobiology, 2000, 72(1): 28–34 A Ruthenium Probe for Cell Viability Measurement Using Flow Cytomet Confocal Microscopy and Time-resolved Luminescence ¶ M. Emilia Jime´ nez-Herna´ ndez 1 , Guillermo Orellana* 1 , Francisco Montero 2 and M. Teresa Portole´ s 2 1 Laboratory of Applied Photochemistry, Department of Organic Chemistry, Faculty of Chemistry, Universidad Complutense de Madrid, Spain and 2 Department of Biochemistry and Molecular Biology I, Faculty of Chemistry, Universidad Complutense de Madrid, Spain Received 27 January 2000; accepted 14 April 2000 ABSTRACT The capability of the new luminescent probe (dibenzo[h,j] dipyrido[3,2-a:2 9 ,3 9 -c]phenazine)bis(2,2 9 -bipyridine)ruthe- nium(II) dication, (RB2Z), to discriminate live and dead cells has been tested on rat hepatocytes and mouse lym- phocytes. RB2Z-stained cells were analyzed using flow cytometry, fluorescence (confocal) microscopy and time- resolved luminescence measurements. The established vi- ability probes propidium iodide (PI) and SYTOX t 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 fluo- rescence 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 m s in presence of fully viable and dead cells, respectively). This ruthenium complex is photosta- ble under laser sources and its 200 nm Stokes shift fa- cilitates multicolor labeling experiments in flow cytome- try and fluorescence microscopy. Unlike the currently available probes, the long-lived excited state of RB2Z also allows assays based on luminescence decay measure- ments. INTRODUCTION Viability assays of both cultured and suspended cells are often used to study their sensitivity to chemical, physical or biological agents. A rapid quantification of bacterial viability is also important to study the movement of pathogens and to preventbacterial colonization or biofilm formation in drinking water reservoirs and refrigeration towers. More- over,dead cells are an important source of interference in flow cytometry that mustbe eliminated from the measure- ments. Notsurprisingly, a large number of tests have been developed to assess cell viability. Different criteria such as the number of surviving cells after addition of the cytotoxic ¶Posted on the web on 17 May 2000. *To whom correspondence should be addressed at: Department of Organic Chemistry, Faculty of Chemistry, Universidad Complu- tense de Madrid, E-28040 Madrid, Spain. Fax: 34-91-3944103; e- mail: orellana@eucmax.sim.ucm.es agent (plate count method), the reproductive potential of th cells,the incorporation of [ 3 H]-thymidine, the intracellular enzymatic activity or the integrity of the plasma membrane are used (1). The popularity of methods based on membrane integrity is due to their versatility, availability, rapidity, safe ty, reproducibility and a direct relationship to cell death. Dy exclusion assays, such as the trypan blue test, allow a direc determination of the percentage of cells in the sample with compromised plasma membrane as a consequence of death However, this colorimetric test must be completed within 3 5 min because the number of stained cells slowly increases after addition of the dye (2). Fluorescent probes have found countless biological, clin- ical and biomedical applications, due to their large sensitiv- ity and selectivity for instrumental measurements compared to colorimetric methods. In this way, several techniques to determine cell viability based on the fluorescence of organic dyes have been employed. These viability measurements ca be made using conventional cuvette or plate fluorometry, flow cytometry and fluorescence microscopy to evaluate the selectively stained living or dead cells. The variety of cell types and their environments makes it difficulto design a universal viability test; therefore, several methods have bee developed for this purpose. A traditional assay to mark cells with intact plasma mem- brane (viable cells) uses fluorescein diacetate (FDA)† (3). Both live and dead populations internalize the diester but only the former retains the resulting fluorescein generated upon hydrolysis by cytoplasmatic esterases. The mostuti- lized fluorescent markers of dead cells are polar DNA-bind- ing dyes, unable to penetrate intact plasma membranes. The high DNA concentration in the cells, and the strong enhanc mentof the probe emission upon binding to the polynucle- otide,render such molecular probes ideal candidates to as- sess cell viability. Widespread markers are the ethidium and propidium iodide(PI) cationsand their homodimers , SYTOX t green or em em ( l ø 615 nm) (SG,l ø 525 nm) max max monomeric and dimeric cyanines such as YOYO t , TOTO t , †Abbreviations: bpy, 2,2 9 -bipyridine; DAPI,4 9 ,6-diamidino-2-phen- ylindole; ddz,dibenzo[h,j]dipyrido[3,2-a:2 9 ,3 9 -c]phenazine; FDA, fluorescein diacetate; HEPES,4-(2-hydroxyethyl)-1-piperazine- ethanesulfonic acid; KR, Krebs–Ringer; PI, propidium iodide; RB2 ( d i b e n z o [ h , j ] d i p y r i d o [ 3 , 2 - a : 2 9 , 3 9 - c ] p h e n a z i n e ) b i s ( 2 , 2 9 - bipyridine)ruthenium(II); SG, SYTOX t green.