A fast kinetic method for assessing mitochondrial membrane potential in isolated hepatocytes with rhodamine 123 and flow cytometry

Abstract

Rhodamine 123 (Rh123) is widely used as a flow cytometric probe for mitochondrial membrane potential (MMP) in metabolic, pharmacologic, and toxicological studies. However, the use of relatively high concentrations of Rh123 (up to 10 pg/ml) and prolonged incubation times (up to 1 h), including washing steps, may be inconvenient for certain applications in which labile cells are used or which demand rapid or repeated analysis. In this paper we describe a rapid kinetic assay of MMP in isolated rat hepatocytes, based upon the quantitation of the initial rate of Rh123 uptake by living cells, selected by their scattering properties. The results indicate that at an appropriate dye-to-cell ratio (in our experiments, 50 ng Rh123/ml for 250,000300,000 cells/ml), the initial rate of Rh123 uptake is a highly reproducible and sensitive parameter for estimation of MMP, as demonstrated by the effects of substrates and inhibitors of the glycolytic pathway and mitochondrial respiration. Because of its simplicity, rapidity (about 5 min) and metabolic implications, this assay would be also suitable for the routine evaluation of metabolic state of cell suspensions, as a complementary test to the standard dualstaining tests of viability. Other possible applications in screening pharmacologic and toxicological analysis are discussed. o 1994 Wiley-Liss, Ine.Key terms: Mitochondria, mitochondrial membrane potential, rhodamine 123, flow cytometry, hepatocyte, glucose metabolism Mitochondrial membrane potential (MMP) is a key parameter to assess cellular energy metabolism under physiological and pathological conditions (5). However, the conventional approach of studying isolated mitochondria in vitro does not allow integration of the mitochondrial function into the whole cell.Flow cytometry provides convenient methods for the analysis of MMP in situ. Recently, most flow cytometric studies on mitochondrial function have been performed with rhodamine 123 (Rh123), a lipophilic cationic fluorochrome which is incorporated by mitochondria according to transmembrane potential (6,9,11,14,20,22). Rh123 has been applied extensively in flow cytometric studies of mitochondrial metabolism (2,30,33) and metabolic heterogeneity (341, cell activation (81, differentiation (71, oncogenic transformation (32), stem cell characterization and purification (37,381, aging (16), and apoptotic death (lo), as well as for assessment of cytotoxicity (20,22,23,24) and drug resistance (15,17). Some of the multiple applications of Rh123 have been reviewed by Ronot et al. (26,271 and Chen (5,6).The most usual techniques to study MMP by flow cytometry in whole cells follow the one described by Johnson et al. (13,14), with slight modifications. Following a 30-60 min equilibration of cells with Rh123

Assessment of mitochondrial membrane potential in yeast cell populations by flow cytometry

“…The data obtained with heat-killed yeast suspensions suggest that Rh123 can be used as a probe to discriminate live from dead cells. This is similar to what has been reported by Juan et al (1994) with animal cells. Actually, heat-killed yeast cells showed a diffuse and more intense fluorescence than untreated cells.…”

“…This confirmed that Rh123 fluorescence changes reflect changes of ∆Ψm, as has been described in intact animal cells (Juan et al, 1994). It has been discussed in previous work that alterations of ∆Ψm may not be the only cause of Rh123 fluorescence changes.…”

“…More recently, Juan et al (1994) used a Rh123 concentration of 250 nM to assess ∆Ψm in mouse liver cells. The Rh123 concentration selected in the present work (50 nM) is much lower than those described in the literature.…”

“…Among the different fluorescent dyes available, rhodamine 123 (Rh123) is the one most frequently used for the assessment of ∆Ψm in mammalian cells (Emaus et al, 1986 ;Johnson et al, 1981 ;Goldstein & Korczac, 1981 ;O'Connor et al, 1988 ;Petit et al, 1990 ;Juan et al, 1994). Rh123 stains mitochondria directly, without passage through endocytotic vesicles and lysosomes , and distributes electrophoretically into the mitochondrial matrix in response to ∆Ψm (Emaus et al, 1986).…”

“…However, depending on the concentration and the cell type, it can also be cytotoxic. So far Rh123 has been used at concentrations up to 10 µg ml −" (Emaus et al, 1986 ;Johnson et al, 1981 ;Goldstein & Korczac, 1981 ;O'Connor et al, 1988 ;Petit et al, 1990 ;Juan et al, 1994 ;Lloyd et al, 1996).…”