Centro de Ciências do Ambiente Departamento de Biologia, Universidade do Minho, Campus de Gualtar, 4719-057 Braga Codex, Portugal1
Laboratório de Citometria, Instituto de Patologia e Imunologia Molecular da Universidade do Porto, Porto, Portugal2
Author for correspondence: Manuela Côrte-Real. Tel: +351 253 604314. Fax: +351 253 678980. e-mail: mcortereal{at}bio.uminho.pt
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ABSTRACT |
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Keywords: mitochondrial functionality, flow cytometry, rhodamine 123, whole yeast cells
Abbreviations: EFM, epifluorescence microscopy; FCM, flow cytometric microscopy; FS, forward scatter; R, ratio; Rh123, rhodamine 123; SS, side scatter
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INTRODUCTION |
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In recent years, many probes for monitoring m have been developed and described with increasing emphasis on membrane-permeable lipophilic cations. Mitochondria are the only organelles known to have a significant membrane potential, with a negative charge inside. Hence lipophilic compounds with delocalized positive charge may be accumulated by mitochondria to a much greater extent than by other organelles (Grinius et al., 1970
). These compounds can freely enter the cell by simple diffusion and although plasma membrane potential contributes to some accumulation into the cytoplasm, the dye concentration there would be much lower than that in the mitochondria. In fact, the Nernst equation predicts a
p of -60 mV and a
m of -180 mV with accumulation ratios of 10 and 1000 in the cytoplasm and mitochondria, respectively. Therefore, the probe concentration inside mitochondria will be 10000-fold higher than that in the extracellular medium (Chen, 1988
).
Among the different fluorescent dyes available, rhodamine 123 (Rh123) is the one most frequently used for the assessment of m in mammalian cells (Emaus et al., 1986
; Johnson et al., 1981
; Goldstein & Korczac, 1981
; OConnor et al., 1988
; Petit et al., 1990
; Juan et al., 1994
). Rh123 stains mitochondria directly, without passage through endocytotic vesicles and lysosomes (Chen et al., 1981
), and distributes electrophoretically into the mitochondrial matrix in response to
m (Emaus et al., 1986
). In yeast the use of that probe has been restricted to the evaluation of the mitochondrial (respiratory) function (Skowronek et al., 1990
; Lloyd et al., 1996
; Lloyd, 1999
) and the detection of efflux pumps (Prudêncio et al., 2000
; Kolaczkowski et al., 1996
). The success of this cationic dye in probing
m in mammalian living cells is closely related to its lower toxicity than other fluorescent cations. 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-1 (Emaus et al., 1986
; Johnson et al., 1981
; Goldstein & Korczac, 1981
; OConnor et al., 1988
; Petit et al., 1990
; Juan et al., 1994
; Lloyd et al., 1996
).
The aim of the present work was to optimize a Rh123 staining protocol to assess qualitative changes of m in whole yeast cells by flow cytometry. Flow cytometry provides a sophisticated technique for research on heterogeneous microbial populations to determine physiological characteristics on a single-cell basis.
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METHODS |
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Fluorochrome solutions.
Concentrated stock solutions of Rh123 (500 µM; Molecular Probes) were prepared in 100% ethanol and stored at -20 °C. Working solutions (25 µM) were prepared by diluting the stock solutions in 100% ethanol and kept on ice in the dark to minimize degradation.
Preparation of yeast suspensions, flow cytometric (FCM) and epifluorescence microscopy (EFM) analysis
. In control assays, cell suspensions were prepared from cultures at early and late exponential phase. Cells were harvested, centrifuged (1600 g for 4 min at 4 °C) and washed twice with ice-cold distilled water, then resuspended (about 1x106 cells ml-1) in sterile commercial water (PARACÉLSIA), pH 6·0. It is generally accepted that this cell concentration allows an adequate data acquisition by flow cytometry. Control suspensions of killed cells were prepared by boiling the cell suspensions, prepared as above, for 10 min.
The Rh123 staining protocol was developed as described in Results. FCM analysis was performed on a EPICS XL-MCL (Beckman-Coulter) flow cytometer, equipped with an argon-ion laser emitting a 488 nm beam at 15 mW. The green fluorescence was collected through a 488 nm blocking filter, a 550 nm long-pass dichroic and a 525 nm band-pass. Twenty thousand cells per sample were analysed. An acquisition protocol was defined to measure forward scatter (FS), side scatter (SS) and green fluorescence (FL1) on a four decades logarithmic scale. Green fluorescence was gated in a scattergram of log SSxlog FS in order to include the subpopulation with the highest frequency and homogeneity in the fluorescence measurements. The data were analysed with the Multigraph software included in the system II acquisition software for the EPICS XL/XL-MCL version 1.0. The ratios between green fluorescence and FS were performed offline with Multiparameter Data Analysis Software, MULTIPLUS AV (Phoenix Flow Systems).
The kinetics of Rh123 uptake by the yeast cells was followed by flow cytometry. For this, green fluorescence acquisition over time of an unstained yeast cell suspension, was started and paused after 1 min. As quickly as possible, Rh123 was added to the cell suspension; the acquisition continued again and was followed for 30 min.
The EFM analyses were performed on a Leitz Laborlux S epifluorescence microscope equipped with a 50 W mercury lamp and a filter set (excitation filter BP 450490, a beam splitter FT510 and an emission filter LP520). Samples of stained cell suspensions (20 µl) were placed between a slide and a cover slip after mixing with an equal volume of antifading reagent (Vectashield Mounting Medium for fluorescence H-1000; Vector Laboratories). The digital images were acquired with a 3CCD Colour Video Camera (SONY, DXC-9100P), a frame grabber (IMAGRAPH, IMASCAN/Chroma-P) and software for image archival and management (HOSPITRANS, Fotoscope version 1.0). In the EFM analysis the concentration of Rh123 used was 500 nM instead of 50 nM, due to the lower sensitivity of EFM compared with FCM. However, the dye to cell concentration ratio was the same as that used in FCM analysis.
Cytotoxicity assays of Rh123.
Cytotoxicity of Rh123 was determined by estimating its effect on cell viability assessed by the plate count method. The c.f.u. were counted after 2 d incubation at 26 °C on YEPD agar plates.
Modification of mitochondrial membrane potential.
To induce a decrease in the mitochondrial membrane potential (m), cells were treated for 10 min at room temperature prior to the Rh123 staining, with one of the following chemical agents (all from Sigma): the uncoupler of oxidative phosphorylation carbonyl cyanide p-(tri-fluoromethoxy)phenyl-hydrazone (FCCP; 5, 10 and 15 µM), the electron-transport inhibitor sodium azide (NaN3; 20 mM) or the potassium ionophore, valinomycin (8 µM). A decrease of
m was also achieved by cell starvation. For this purpose, cells from an overnight culture, prepared as described above, were resuspended in culture medium without glucose and incubated for 48 h in a water bath, with magnetic stirring (250 r.p.m.), at 26 °C.
Incubation of the cells with 2-deoxy-D-glucose (2-DOG; Sigma) was also carried out. This compound is a glucose analogue that competitively inhibits glucose transport and depletes cells of ATP. Cells were also incubated with 2-DOG (100 mM) at room temperature, for 10 min before Rh123 staining.
The m was also evaluated in the presence of other agents that disrupt mitochondrial metabolism, namely oligomycin (0·4 µg ml-1), an F0-ATP-synthase inhibitor, and nigericin (5 µM), a dissipater of
pH (both from Sigma). These agents were added 10 min before Rh123 staining.
Stimulation of m was achieved by incubation at room temperature with 100 or 150 mM glucose, or 5 mM succinate, 10 min before Rh123 staining. Incubation with glucose was carried out at pH 6·0 and incubation with succinate at pH 3·0. Both substrates were from Merck.
Reproducibility of the results.
All experiments were repeated at least three times. The data reported are from one representative experiment.
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RESULTS |
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Normalized Rh123 fluorescence
The analysis of biparametric histograms, plotting log SS against log FS, of cell suspensions of Zygosaccharomyces bailii or Saccharomyces cerevisiae revealed a marked heterogeneity, in both relative complexity and size. In the scattergram of a Z. bailii cell suspension (Fig. 1a) several subpopulations can be visualized. The subpopulation at the top right consists mainly of cell aggregates, since it disappeared after a brief ultrasonication of the cell suspension (data not shown). The subpopulation with the lowest scatter most probably corresponds to young cells that have been recently separated from their mother cells. The most numerous subpopulation is present in the middle of the scattergram. The monoparametric histogram of the total fluorescence of this subpopulation has a great coefficient of variation (Fig. 1b
), and three different gated regions (A, B and C) can be defined in the corresponding biparametric histogram (Fig. 1a
). Previous results on DNA distributions (Howlett & Avery, 1999
; Fortuna et al., 2000
) showed that these regions are related to the different phases of the cell cycle; the A, B and C gated regions correspond to cells in phases G0/G1, S and G2/M, respectively. The fluorescence obtained in each gated region gives rise to three different distributions (Fig. 1c
), pointing to the necessity to normalize the Rh123 fluorescence. Actually, cells with the same fluorochrome concentration but different relative volumes showed different levels of fluorescence. Normalization was achieved by representing in a monoparametric histogram the log of the green Rh123 fluorescence intensity, divided by the log of the FS signal. This ratio (R) was calculated by MULTIPLUS software (Phoenix Flow Systems).
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Following these results, the Rh123 concentration selected was 50 nM rather than 25 or 10 nM. The former concentration value, although yielding a cell fluorescence level distinct from autofluorescence, would allow a more sensitive detection of decreases in the mean fluorescence intensity and hence in m. Moreover, 50 nM Rh123 was not cytotoxic for either of the yeast species tested when assessed by counts of c.f.u. (data not shown).
The kinetics of Rh123 uptake by S. cerevisiae and Z. bailii was studied by FCM (data not shown). For both yeast species the uptake of the probe was very fast and the steady level of fluorescence was reached in about 10 min. Rh123 uptake was also followed by EFM (Fig. 4). By observation of the image obtained after 5 min incubation it was concluded that the observed rapid initial uptake corresponded to non-specific binding of the dye to the cell envelope. However, after 10 min Rh123 was electrophoretically accumulated into the mitochondria and gave a specific staining of this organelle. The non-specific binding of the dye, associated with the cell envelope, still observed at this incubation time is most probably negligible for the dye concentration used for FCM.
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The mean fluorescence intensity of cells stained with Rh123 at room temperature or at 37 °C was identical. Therefore, incubation at room temperature was selected.
Most of the buffers used in staining media have a complex ionic composition and different pH values that could directly interfere with Rh123 uptake. However, similar levels of fluorescence were observed (data not shown) for yeast cell suspensions from both species stained with Rh123 at pH 3·0 or 6·0, indicating that the mitochondrial accumulation of the dye is independent of the extracellular pH. Furthermore, based on the dependence of the plasma membrane potential (p) on the extracellular pH (van den Broeck, 1982
), we concluded that mitochondrial accumulation of Rh123, under the optimized staining conditions is hardly affected by
p.
To evaluate the influence of the medium composition on Rh123 staining, the fluorescence intensity of cells stained in mineral medium (pH 4·5, MGV medium), PBS (pH 7·0) or sterile water (pH 6·0) was compared. Higher levels of fluorescence were achieved when staining with Rh123 was carried out in sterile water (Fig. 5).
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Definition of a simple staining protocol with Rh123
In summary, the conditions selected for the optimal staining with Rh123 and for assessment of mitochondrial membrane potential included the use of: (i) yeast cell suspensions in sterile water at a final cell concentration of 1x106 cells ml-1; (ii) Rh123 at a final concentration of 10 nM for late exponential phase S. cerevisiae cells, and 50 nM for early exponential phase S. cerevisiae cells and Z. bailii, independent of the growth phase; (iii) staining for 10 min at room temperature in the dark.
Validation of the staining protocol with Rh123 for the assessment of m
To ascertain whether Rh123 accumulation in whole yeast cells was correlated to m, the effects of ionophores, specific inhibitors and substrates of mitochondrial respiration were tested. Valinomycin and FCCP [carbonyl cyanide p-(trifluoromethoxy)phenyl-hydrazone] at the concentrations tested had no effect on Rh123 accumulation in yeast cells (data not shown). The same result was obtained by other authors and attributed to the possible formation of complexes between putative adducts and components of the inner mitochondrial membrane. Some of these complexes are freely accessible whatever the energy status of mitochondria but others are hidden in the energized state and freely accessible only in the de-energized state of the organelle (Salvioli et al., 1997
).
Cells exposed to sodium azide or 2-deoxy-D-glucose, or starved cells displayed a decrease in the Rh123 accumulation compatible with a decrease in m (Fig. 6
). Furthermore, the two former conditions did not affect the dye accumulation in heat-killed yeast cells (data not shown). Yeast cells treated with oligomycin and nigericin showed increased Rh123 staining (data not shown). These results are consistent with previous reports by other authors. Actually, oligomycin was used by Macouillard-Poulletier de Gannes et al. (1998)
to mimic a decrease in cellular ATP demand (transition to state 4). In a similar way, these authors observed an increase in
m linked to the inhibition of ATP synthase by oligomycin associated with a significant increase in DiOC6(3) fluorescence.
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The addition of succinate, which provides reducing equivalents to the respiratory chain through succinate dehydrogenase, increased the accumulation of Rh123 in living cells.
Strain J-1-3 of S. cerevisiae is a null mutant for the ADP/ATP translocator and compared with the parental strain, produced a small but significant decrease of m when assessed by Rh123 staining. Incubation of this mutant strain with sodium azide should abolish the mitochondrial respiratory function:
m should collapse and Rh123 should not accumulate in the mitochondria. Actually, under these conditions the J-1-3 mutant strain displayed a mean fluorescence intensity quite similar to the autofluorescence (Fig. 7
).
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DISCUSSION |
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Most of the methods recently described for the assessment of m were derived from the protocol of Johnson et al. (1980)
, in which the Rh123 concentration used was 25 µM. More recently, Juan et al. (1994)
used a Rh123 concentration of 250 nM to assess
m in mouse liver cells. The Rh123 concentration selected in the present work (50 nM) is much lower than those described in the literature. At this concentration the cytotoxic effects of the probe and its non-specific binding to other intracellular organelles were eliminated or at least became negligible. The results obtained with the ADP/ATP null mutant strain of S. cerevisiae in the presence of sodium azide further confirmed this. In fact, in this strain under conditions where
m is collapsed, Rh123 accumulation in the mitochondria was prevented and the level of staining corresponded almost to that of autofluorescence.
In both S. cerevisiae and Z. bailii, comparable levels of fluorescence intensity were obtained regardless of the incubation temperature (room temperature or 37 °C). In addition, and consistent with the results reported by other authors (Ronot et al., 1986 ), the uptake of Rh123 (50 nM) at room temperature is rapid and equilibrium is reached after 10 min.
In the two yeast species tested under the optimized staining conditions, an increase or decrease in the R value of stained yeast cell suspensions was observed after treatment with hyperpolarizing (substrate of mitochondrial respiration) or depolarizing (ionophores or specific inhibitors of respiration) agents, respectively. This confirmed that Rh123 fluorescence changes reflect changes of m, as has been described in intact animal cells (Juan et al., 1994
).
It has been discussed in previous work that alterations of m may not be the only cause of Rh123 fluorescence changes. In fact, changes in mitochondrial Rh123 accumulation can be independent of
m and reflect changes in either mitochondrial mass or the cytoplasmic concentration of the probe. An example of the former situation has been reported for an ADP/ATP translocator mutant (Petit et al., 1996
). Such a mutant has a nearly normal
m; however, the decrease observed in
m by Rh123 staining is associated with a decrease in mitochondrial mass, assessed by cardiolipin staining (by nonyl acridine orange dye). This aspect further reinforces the need to complement
m studies with a mitochondrial structure probe when changes in the cellular content of mitochondria are suspected to occur. Moreover, alterations of the Rh123 cytoplasmic concentration might cause fluorescence alterations independently of
m (Bernardi et al., 2001
). These alterations can be elicited either by the activity of multidrug resistance pumps for Rh123 or changes in the plasma membrane potential (
p). Elimination of the interference of active efflux pumps on
m determinations by the optimized Rh123 staining protocol is difficult since it would imply the knowledge for all the active systems, that use Rh123 as substrate, of the respective affinity constants. Actually, these data are only available in S. cerevisiae, and there only for specific active efflux systems (Kolaczkowski et al., 1996
). That mitochondrial accumulation of Rh123 was independent of the extracellular pH eliminates
p interference on
m determinations.
Salvioli et al. (2000) observed that in a given cell only some mitochondria were polarized, indicating heterogeneous behaviour at the mitochondrial level. If observed in yeast, this further complicates
m measurements.
The different Rh123 concentrations required to stain optimally S. cerevisiae glucose-grown cells from early or late-exponential phase is most probably due to the well known shift from fermentative to respiratory metabolism (Pronk et al., 1996 ), and to inherent and significant changes in the mitochondrial compartment (Visser, 1995
) associated with the growth phase. The same is not found in Z. bailii. This observation is consistent with the higher relative proportion of respiration to fermentation (1:3) in Z. bailii (Fernandes et al., 1997
) as compared with S. cerevisiae (1:20) (Lagunas, 1986
). In addition, and in contrast to the pro-mitochondria described in glucose-grown S. cerevisiae cells, Z. bailii displayed a higher proportion of mature mitochondria (P.Ludovico, F. Sansonetty, M. T. Silva, & M. Côrte-Real, unpublished results). The results referred to above indicate that the application of the Rh123 staining protocol to other species should take into account possible differences or changes in the mass of the mitochondrial compartment with growth conditions.
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. It should be expected that in the former cells, Rh123 staining would produce a lower fluorescence level. A possible explanation could be attributed to the loss of mitochondrial membrane integrity in the dead cells and consequently to a non-localized distribution of Rh123. The observed behaviour might be determined by different fluorescence-quenching effects of the probe when interacting with the denatured and precipitated cell constituents.
It has been recently reported that S. cerevisiae becomes committed to a programmed cell death process in response to acetic acid (Ludovico et al., 2001 ). The protocol described here will be crucial in the further evaluation of the involvement of mitochondrial function, namely of
m changes in triggering such active cell death processes.
Presently one of the most generalized probes to assess mitochondrial membrane potential in mammalian cells is JC-1 (Seligmann & Gallin, 1986 ; Cossarizza et al., 1993
). In the presence of a high
m, JC-1 forms J-aggregates that are associated with a large shift in the wavelength of fluorescence emission. However, one disadvantage of JC-1 compared with Rh123 is related to the speed of its responses to
m changes, which are slower than those seen under equivalent conditions when using Rh123 (Haugland, 1996
). This suggests that the slower redistribution of JC-1 could result in an underestimation of the actual rate of change of
m.
In summary, optimization of the Rh123 staining conditions described here allowed the definition of a flow cytometric protocol for the qualitative assessment of rapid changes in m of yeasts. However, it should be emphasized that when the Rh123 staining protocol is to be applied to a new yeast species, preliminary experiments should be carried out to establish the optimal staining conditions.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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van den Broeck, P. (1982). The energetics of sugar transport in yeast. PhD thesis, University of Leiden, The Netherlands.
Chen, L. B. (1988). Mitochondrial membrane potential in living cells. Annu Rev Cell Dev Biol 4, 155-181.
Chen, L. B. (1989). Fluorescent labeling of mitochondria. Methods Cell Biol 29, 103-203.[Medline]
Chen, L. B., Summerhayes, I. C., Johnson, L. V., Walsh, M. L., Bernal, S. D. & Lampidis, T. J. (1981). Probing mitochondria in living cells with rhodamine 123. Cold Spring Harbor Symp Quant Biol 46, 141-155.
Cossarizza, A., Baccarani-Contri, M., Kalashnikova, G. & Franceshi, C. (1993). A new method for the cytofluorimetric analysis of mitochondrial membrane potential using the J-aggregate forming lipophilic cation 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolcarbocyanine iodide (JC-1). Biochem Biophys Res Commun 197, 40-45.[Medline]
Emaus, R. K., Grunwald, R. & Lemasters, J. (1986). Rhodamine 123 as a probe of transmembrane potential in isolated rat-liver mitochondria: spectral and metabolic properties. Biochim Biophys Acta 850, 436-448.[Medline]
Fernandes, L., Cõrte-Real, M., Loureiro, V., Loureiro-Dias, M. C. & Leão, C. (1997). Glucose respiration and fermentation in Zygosaccharomyces bailii and Saccharomyces cerevisiae express different sensitivity patterns to ethanol and acetic acid. Lett Appl Microbiol 25, 249-253.[Medline]
Fortuna, M., Sousa, M. J., Cõrte-Real, M., Leão, C., Salvador, A. & Sansonetty, F. (2000). Cell cycle analysis of yeasts using Syber Green I. In Current Protocols in Cytometry, pp. 11.13.111.13.9. Edited by J. P. Robinson. New York: Wiley.
Goldstein, S. D. & Korczac, L. B. (1981). Status of mitochondria in living fibroblasts during growth and senescence in vitro: use of the laser dye rhodamine 123. J Cell Biol 91, 392-398.[Abstract]
Grinius, L. L., Jasaitis, A. A., Kadziaukas, Y. P., Liberman, E. A., Skulachev, V. P., Topaly, V. P., Tsofina, L. M. & Vladimirova, M. A. (1970). Conversion of biomembrane-produced energy into electric form. I. Submitochondrial particles. Biochim Biophys Acta 216, 1-12.[Medline]
Haugland, R. P. (1996). Handbook of fluorescent probes. Eugene, OR: Molecular Probes.
Howlett, N. G. & Avery, S. V. (1999). Flow cytometric investigation of heterogeneous copper-sensitivity in asynchronously grown Saccharomyces cerevisiae. FEMS Microbiol Lett 176, 379-386.[Medline]
Johnson, L. V., Walsh, M. L. & Chen, L. B. (1980). Localization of mitochondria in living cells with rhodamine 123. Proc Natl Acad Sci 2, 990-994.
Johnson, L. V., Walsh, M. L., Bokus, B. J. & Chen, L. B. (1981). Monitoring of relative mitochondrial membrane potential in living cells by fluorescence microscopy. J Cell Biol 83, 526-535.
Juan, G., Cavazzoni, M., Sáez, G. T. & OConnor, J. E. (1994). A fast kinetic method for assessing mitochondrial membrane potential in isolated hepatocytes with rhodamine 123 and flow cytometry. Cytometry 15, 335-342.[Medline]
Kolaczkowski, M., van der Rest, M., Cybularz-Kolaczkowska, A., Soumillion, J. P., Konings, W. & Goffeau, A. (1996). Anticancer drugs, ionophoric peptides, and steroids as substrates of the yeast multidrug transporter Pdr5p. J Biol Chem 271, 31543-31548.
Lagunas, R. (1986). Misconceptions about the energy metabolism of Saccharomyces cerevisiae. Yeast 2, 221-228.[Medline]
Lloyd, D. (1999). Evaluating the mitochondrial (respiratory) function of yeast. Current Protocols in Cytometry. p. 11.10.4. Edited by J. P. Robinson. New York: Wiley.
Lloyd, D., Moran, C. A., Suller, M. T. E., Dinsdale, M. G. & Hayes, H. J. (1996). Flow cytometric monitoring of rhodamine 123 and a cyanine dye uptake by yeast during cider fermentation. J Inst Brew 102, 251-259.
Ludovico, P., Sousa, M. J., Silva, M. T., Leão, C. & Cõrte-Real, M. (2001). Saccharomyces cerevisiae commits to a programmed cell death process in response to acetic acid. Microbiology 147, 2409-2415.
Macouillard-Poulletier de Gannes, F., Belaud-Rotureau, M. A., Voisin, P., Leducq, N., Belloc, F., Canioni, P. & Diolez, P. (1998). Flow cytometric analysis of mitochondrial activity in situ: application to acetylceramide-induced mitochondrial swelling and apoptosis. Cytometry 33, 333-339.[Medline]
Nicholls, D. G. (1982). Bioenergetics: an Introduction to the Chemiosmotic Theory. London & New York: Academic Press.
OConnor, J. E., Vargas, J. L., Kimler, B. F., Hernandez-Yago, J. & Grisolia, S. (1988). Use of rhodamine 123 to investigate alterations in mitochondrial activity in isolated mouse liver mitochondria. Biochem Biophys Res Commun 151, 568-573.[Medline]
Petit, P. X., OConnor, J. E., Grunwald, D. & Brown, S. C. (1990). Analysis of the membrane potential of rat- and mouse-liver mitochondria by flow cytometry and possible applications. Eur J Biochem 194, 389-397.[Abstract]
Petit, P. X., Glad, N., Marie, D., Kieffer, H. & Métézeau, P. (1996). Discrimination of respiratory dysfunction in yeast mutants by confocal microscopy, image, and flow cytometry. Cytometry 23, 28-38.[Medline]
Pronk, J. T., Steensma, H. Y. & Dijken, J. P. V. (1996). Pyruvate metabolism in Saccharomyces cerevisiae. Yeast 12, 1607-1633.[Medline]
Prudncio, C., Sansonetty, F., Sousa, M. J., Cõrte-Real, M. & Leão, C. (2000). Rapid detection of efflux pumps and their relation with drug resistance in yeast cells. Cytometry 39, 26-35.[Medline]
Ronot, X., Benel, L., Adolphe, M. & Mounolou, J. C. (1986). Mitochondrial analysis in living cells: the use of rhodamine 123 and flow cytometry. Biol Cell 57, 1-8.[Medline]
Salvioli, S., Ardizzoni, A., Franceschi, C. & Cossarizza, A. (1997). JC-1, but not DiOC6(3) or rhodamine 123, is a reliable fluorescent probe to assess changes in intact cells: implications for studies on mitochondrial functionality during apoptosis. FEBS Lett 411, 77-82.[Medline]
Salvioli, S., Dobrucki, J., Moretti, L., Troiano, L., Fernandez, M., Pinti, M., Pedrazzi, J., Franceschi, C. & Cossarizza, A. (2000). Mitochondrial heterogeneity during staurosporine-induced apoptosis in HL60 cells: analysis at the single cell and single organelle level. Cytometry 40, 189-197.[Medline]
Seligmann, B. E. & Gallin, J. I. (1986). Comparison of indirect probes of membrane potential utilized in studies of human neutrophils. J Cell Physiol 105, 105-115.
Shapiro, H. M. (1994). Cell membrane potential analysis. Methods Cell Biol 41, 121-133.[Medline]
Skowronek, P., Krummeck, G., Haferkamp, O. & Rodel, G. (1990). Flow cytometry as a tool to discriminate respiratory-competent and respiratory-deficient yeast cells. Curr Genet 18, 265-267.[Medline]
van Uden, N. (1967). Transport-limited fermentation and growth of Saccharomyces cerevisiae and its competitive inhibition. Arch Mikrobiol 58, 155-168.[Medline]
Visser, W. (1995). Oxygen requirements of fermentative yeasts. PhD thesis, Technical University of Delft.
Wium, H., Malfeito-Ferreira, M., Loureiro, V. & Aubyn, A. (1990). A rapid characterization of yeast contaminants associated with sparkling wine production. Industrie Bevande 19, 504-506.
Received 1 June 2001;
revised 13 July 2001;
accepted 26 July 2001.