Characterisation of proton fluxes across the cytoplasmic membrane of the yeast Saccharomyces cerevisiae.

We have tested the efficacy of fluorescent probes for the measurement of intracellular pH in Saccharomyces cerevisiae. Of the compounds tested (fluorescein, carboxyseminaphthorhodafluor-1 (C.SNARF-1) and 2',7'bis(carboxyethyl)-5(6')-carboxyfluorescein), C.SNARF-1 was found to be the most useful indicator of internal pH. Fluorescence microscopy showed that in Saccharomyces cerevisiae strain DAUL1, C.SNARF-1 and fluorescein had a heterogeneous distribution, with dye throughout the cytoplasm and concentration of the dye to an area close to the cell membrane. This region was also labeled by quinacrine, which is known to accumulate in acidic regions of the cell. Saccharomyces cerevisiae BJ4932, which carries a defect in vacuolar acidification, did not show the same degree of dye concentration, suggesting that the site of C.SNARF-1 and fluorescein localisation in DAUL1 is the acidic vacuole. Changes in intracellular pH could be monitored by measuring changes in the fluorescence intensity of C.SNARF-1. The addition of glucose caused an initial, rapid decrease in fluorescence intensity, indicating a rise in cellular pH. This was followed by slow acidification. Fluorescence intensity changes were similar in all strains studied, suggesting that the localisation of dye to acidic regions does not affect the measurement of intracellular pH in DAUL1. The changes in intracellular pH on the addition of glucose correlated well with glucose-induced changes in external pH. Preincubation of cells in the presence of the plasma membrane H(+)-ATPase inhibitor diethylstilbestrol reduced extracellular acidification and intracellular alkalinisation on the addition of glucose. Both amiloride and 5-(N-ethyl-N-isopropyl)amiloride also inhibited glucose-induced proton fluxes. Phorbol 12-myristate 13-acetate had no effect on the activity of the plasma membrane ATPase.     

Simultaneous pH Measurement in Endocytic and Cytosolic Compartments in Living Cells using Confocal Microscopy

Intracellular pH is tightly regulated and differences in pH between the cytoplasm and organelles have been reported¹. Regulation of cellular pH is crucial for homeostatic control of physiological processes that include: protein, DNA and RNA synthesis, vesicular trafficking, cell growth and cell division. Alterations in cellular pH homeostasis can lead to detrimental functional changes and promote progression of various diseases². Various methods are available for measuring intracellular pH but very few of these allow simultaneous measurement of pH in the cytoplasm and in organelles. Here, we describe in detail a rapid and accurate method for the simultaneous measurement of cytoplasmic and organellar pH by using confocal microscopy on living cells³. This goal is achieved with the use of two pH-sensing ratiometric dyes that possess selective cellular compartment partitioning. For instance, SNARF-1 is compartmentalized inside the cytoplasm whereas HPTS is compartmentalized inside endosomal/lysosomal organelles. Although HPTS is commonly used as a cytoplasmic pH indicator, this dye can specifically label vesicles along the endosomal-lysosomal pathway after being taken up by pinocytosis^3,4. Using these pH-sensing probes, it is possible to simultaneously measure pH within the endocytic and cytoplasmic compartments. The optimal excitation wavelength of HPTS varies depending on the pH while for SNARF-1, it is the optimal emission wavelength that varies. Following loading with SNARF-1 and HPTS, cells are cultured in different pH-calibrated solutions to construct a pH standard curve for each probe. Cell imaging by confocal microscopy allows elimination of artifacts and background noise. Because of the spectral properties of HPTS, this probe is better suited for measurement of the mildly acidic endosomal compartment or to demonstrate alkalinization of the endosomal/lysosomal organelles. This method simplifies data analysis, improves accuracy of pH measurements and can be used to address fundamental questions related to pH modulation during cell responses to external challenges.     

Improved method for measuring intracellular Ca++ with fluo-3

The accuracy of flow cytometric measurement of intracellular calcium with fluo-3 is compromised by variation in basal fluorescence intensity due to heterogeneity in dye uptake or compartmentalization. We have loaded cells simultaneously with fluo-3 and SNARF-1. When SNARF-1 fluorescence is collected at approximately 600 nm, its intensity does not change upon cell activation. Furthermore, fluo-3 and SNARF-1 fluorescence signals exhibit a linear relationship. The ratio of fluo-3 to SNARF-1 eliminates a significant proportion of variation in fluorescence intensity caused by variation in fluo-3 uptake and thus can be used as a sensitive parameter for measuring changes in [Ca2+]i.     

Use of 1,4 diacetoxy-2,3 dicyanobenzol in microspectrofluorometry for determining the intracellular pH of isolated living cells

Microspectrofluorometry allows to obtain the fluorescence spectrum of an isolated living cell. When cells are preincubated with 1,4 diacetoxy-2,3 dicyanobenzol the cellular fluorescence spectrum can be resolved in its components i.e. the characteristic fluorescence spectrum of each ionized forms of the probe and the intrinsic cell fluorescence spectrum due to NAD(P)H. This allows the determination of the intracellular pH with good accuracy. Furthermore, comparison between the intensity of the intrinsic cell fluorescence and the probe fluorescence intensity offers us an opportunity to monitor the intracellular amount of the drug.     

Intracellular pH distribution in Saccharomyces cerevisiae cell populations, analyzed by flow cytometry

Intracellular pH has an important role in the maintenance of the normal functions of yeast cells. The ability of the cell to maintain this pH homeostasis also in response to environmental changes has gained more and more interest in both basic and applied research. In this study we describe a protocol which allows the rapid determination of the intracellular pH of Saccharomyces cerevisiae cells. The method is based on flow cytometry and employs the pH-dependent fluorescent probe carboxy SNARF-4F. The protocol attempts to minimize the perturbation of the system under study, thus leading to accurate information about the physiological state of the single cell. Moreover, statistical analysis performed on major factors that may influence the final determination supported the validity of the optimized protocol. The protocol was used to investigate the effect of external pH on S. cerevisiae cells incubated in buffer. The results obtained showed that stationary cells are better able than exponentially grown cells to maintain their intracellular pH homeostasis independently of external pH changes. Furthermore, analysis of the intracellular pH distribution within the cell populations highlighted the presence of subpopulations characterized by different intracellular pH values. Notably, a different behavior was observed for exponentially grown and stationary cells in terms of the appearance and development of these subpopulations as a response to a changing external pH.     

Sequential occurrence of mitochondrial and plasma membrane alterations, fluctuations in cellular Ca2+ and pH during initial and later phases of cell death

The sequential occurrence of plasma and mitochondrial membrane alterations, intra-cellular pH shifts and changes in intracellular Ca²⁺ concentration after induction of cell death was monitored by flow cytometry in Jurkat and HSB2-cells. Cell death was induced by treatment with anti-Fas antibodies or by irradiation. Phosphatidylserine (PS) exposure and plasma membrane integrity were measured with FITC-Annexin V adhesion and by Propidium Iodide exclusion. Transition of the mitochondrial membrane potential was monitored by the occurrence of decay of DiOC₆ fluorescence. Intracellular pH shifts were monitored by changes in the ratio of fluorescence at 575 nm and at 635 nm of SNARF-1-AM. Fluctuations in intracellular Ca²⁺ concentration were established by changes in Fura red quenching.The Jurkat cells were sensitive to anti-Fas treatment, while HSB-2 cells were not. HSB-2 cells appeared more sensitive to radiation damage than Jurkat cells.In all experiments the transition of mitochondrial membrane potential occurred first, almost immediately followed by PS exposure. Fluctuations in intracellular Ca²⁺ concentration occurred later and were less outspoken. A decrease in intracellular pH occurred not earlier than 24 hours after anti-Fas treatment. Chelation of intracellular Ca²⁺ concentration with BAPTA-AM had no effect on the time sequence of cell death related events.     

Intracellular pH Distribution in Saccharomyces cerevisiae Cell Populations, Analyzed by Flow Cytometry

Intracellular pH has an important role in the maintenance of the normal functions of yeast cells. The ability of the cell to maintain this pH homeostasis also in response to environmental changes has gained more and more interest in both basic and applied research. In this study we describe a protocol which allows the rapid determination of the intracellular pH of Saccharomyces cerevisiae cells. The method is based on flow cytometry and employs the pH-dependent fluorescent probe carboxy SNARF-4F. The protocol attempts to minimize the perturbation of the system under study, thus leading to accurate information about the physiological state of the single cell. Moreover, statistical analysis performed on major factors that may influence the final determination supported the validity of the optimized protocol. The protocol was used to investigate the effect of external pH on S. cerevisiae cells incubated in buffer. The results obtained showed that stationary cells are better able than exponentially grown cells to maintain their intracellular pH homeostasis independently of external pH changes. Furthermore, analysis of the intracellular pH distribution within the cell populations highlighted the presence of subpopulations characterized by different intracellular pH values. Notably, a different behavior was observed for exponentially grown and stationary cells in terms of the appearance and development of these subpopulations as a response to a changing external pH.     

Chemotactic factor-induced activation of Na+/H+ exchange in human neutrophils. II. Intracellular pH changes

The intracellular pH (pHi) changes resulting from chemotactic factor-induced activation of Na+/H+ exchange in isolated human neutrophils were characterized. Intracellular pH was measured from the equilibrium distribution of [14C]-5,5-dimethyloxazolidine-2,4-dione and from the fluorescence of 6-carboxyfluorescein. Exposure of cells to 0.1 microM N-formyl-methionyl-leucyl-phenylalanine (FMLP) in 140 mM Na+ medium at extracellular pH (pHo) 7.40 led to a rise in pHi along an exponential time course (rate coefficient approximately 0.55 min-1). By 10 min, a new steady-state pHi was reached (7.75-7.80) that was 0.55-0.60 units higher than the resting pHi of control cells (7.20-7.25). The initial rate of H+ efflux from the cells (approximately 15 meq/liter X min), calculated from the intrinsic intracellular buffering power of approximately 50 mM/pH, was comparable to the rate of net Na+ influx (approximately 17 meq/liter X min), an observation consistent with a 1:1 stoichiometry for Na+/H+ exchange. This counter-transport could be inhibited by amiloride (apparent Ki approximately 75 microM). When either the external ([Na+]o) or internal Na ([Na+]i) concentrations, pHo, or pHi were varied independently, the new steady-state [Na+]i and pHi values in FMLP-stimulated cells were those corresponding to a chemical equilibrium distribution of Na+ and H+ across the cell membrane. By analogy to other activated cells, these results indicate that an alkalinization of pHi in human neutrophils is mediated by a chemotactic factor-induced exchange of internal H+ for external Na+.     

Isolation of nuclei from epidermis cells of larvae of the blowfly Calliphora vicina R.-D

Rapid microspectrofluorometry has been used to evaluate 1-pyrene-butyric acid as an oxygen probe in single living EL2 ascites tissue culture cells. Despite instrumental conditions preventing detection of the pyrene butyric acid maxima at 380 and 400 nm, the probe having penetrated the cell can be easily identified (maximum around 440 nm in unconnected spectra) from the fluorescence emission spectrum, as compared with NAD(P)H emission in controls (maximum around 460 nm). Fluorescence changes during gradually increasing anaerobiosis under nitrogen flow, are compatible with a linear relationship between the reciprocal of the fluorescence intensity and the intracellular oxygen concentration (increase in 430, 434, 442/461 nm ratios at anaerobiosis). The cells having absorbed the probe continue to catabolize glycolytic substrate, but some inhibition is noticeable (e.g. from the amplitude of the NAD(P)H fluorescence increase spectrum due to intracellular addition of glucose-6-P). In principle rapid microspectrofluorometry allows a multiprobe (e.g. 1-pyrene-butyric acid for oxygen, vs NAD(P)H for metabolism) exploration of the living cell.     

Dihydrorhodamine 123 is superior to 2,7-dichlorodihydrofluorescein diacetate and dihydrorhodamine 6G in detecting intracellular hydrogen peroxide in tumor cells

Dihydrorhodamine 123 (DHR 123), 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA), and dihydrorhodamine 6G (DHR 6G) were evaluated as probes for detecting cellular hydrogen peroxide levels in SPC-A-1 lung adenocarcinoma cells. Imaging techniques and fluorescence-activated cell scan were used in the study of the probe responses. Obvious green fluorescence was established after a 25-min exposure. After staining with MitoTracker Orange CM-H2TMRos (a probe for mitochondria) and the abovementioned probes simultaneously, only the DHR 123 and DHR 6G groups exhibited legible green fluorescence in the mitochondrial regions. Furthermore, the DHR 6G group exhibited weaker fluorescence intensity. When 100 microM H2O2 was added to SPC-A-1 cells loaded with these probes, the intracellular fluorescence increased rapidly and significantly. Our results suggest that DHR 123 is superior for the instantaneous detection of cellular hydrogen peroxide in SPC-A-1 cells.     

Ratiometric measurement of intracellular pH in cultured human keratinocytes using carboxy-SNARF-1 and flow cytometry

In this study we describe a method to measure intracellular pH in cultured human keratinocytes using flow cytometry. Keratinocytes pose a technical problem because the population is heterogeneous with respect to size and metabolic activity (nonspecific esterase activity), resulting in variability in dye uptake. In order to compensate for this, dyes were selected that change colour with pH. The ratio of fluorescence intensities at two wavelengths was recorded and used as a measure of intracellular pH by reference to the pH in the presence of the proton ionophore nigericin. However, methods published till now do not routinely combine the ratiometric technique and excitation with an argon ion laser set at 488 nm. Therefore we have tested the recently developed pH-sensitive dye carboxyseminaphthorhodafluor-1 (SNARF-1) as a possible candidate for flow cytometric pH measurements and compared it with 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF) and 2,3-dicyanohydroquinone (DCH) with respect to emission spectra, resolution, range, and stability of cellular fluorescence. SNARF-1 had a practical and stable excitation wavelength of 488 nm rather than UV, it offered the possibility of ratiometric measurements on the basis of a real emission shift, and had superior resolution for the pH range 7-8. With SNARF-1 we found that keratinocytes cultured under low serum conditions (0.2%) contain a higher proportion of cells with relatively low intracellular pH compared to high serum cultures (6%). Furthermore, pH changes were followed by changes in relative DNA content. These findings suggest that intracellular pH can be an early functional proliferation marker for human keratinocytes.     

Radiation-induced apoptosis of stem/progenitor cells in human umbilical cord blood is associated with alterations in reactive oxygen and intracellular pH

To investigate the sensitivity of human hematopoietic stem cell populations to radiation and its relevance to intracellular events, specifically alteration in cellular energy production systems, we examined the frequency of apoptotic cells, generation of superoxide anions (O*2-), and changes in cytosol pH in umbilical cord blood (UCB) CD34+/CD38-, CD34+/CD38+ and CD34-/CD38+ cells before and after 5Gy of X-irradiation. Human UCB mononucleated cells were used in this study. After X-irradiation and staining subgroups of the cells with fluorescence (FITC, PE, or CY)-labeled anti-CD34 and anti-CD38 antibodies, analyses were performed by FACScan using as stains 7-amino-actinomycin D (7-AAD) for the detection of apoptosis, and hydroethidine (HE) for the measurement of O*2- generation in the cells. For intracellular pH, image analysis was conducted using confocal laser microscopy after irradiation and staining with carboxy-SNAFR-1. The frequency of apoptotic cells, as determined by cell staining with 7-AAD, was highest in the irradiated CD34+/CD38- cell population, where the level of O*2- detected by the oxidation of HE was also most highly elevated. Intracellular pH measured with carboxy-SNARF-1-AM by image cytometer appeared to be lowest in the same irradiated CD34+/CD38- cell population, and this intracellular pH decreased as early as 4 h post-irradiation, virtually simultaneous with the significant elevation of O*2- generation. These results suggest that the CD34+/CD38- stem cell population is sensitive to radiation-induced apoptosis as well as production of intracellular O*2-, compare to more differentiated CD34+/CD38+ and CD34-/CD38+ cells and that its intracellular pH declines at an early phase in the apoptosis process.     

Fluorescence emission spectroscopy of 1,4-dihydroxyphthalonitrile. A method for determining intracellular pH in cultured cells.

We have developed new methodology for measuring intracellular pH (pHi) in cultured cell monolayers and epithelia by analyzing the emission spectra of the trapped fluorescent pH probe, 1,4-dihydroxyphthalonitrile (1,4-DHPN). This compound is unique since both its acid and base forms possess different fluorescence emission characteristics that can be used to quantitate pHi. The fluorescence difference spectrum between an acid and alkaline solution of 1,4-DHPN has a maximum at 455 nm and a minimum at 512 nm. By determining the ratio of the intensity at these two wavelengths as a function of pH, a calibration curve was constructed. Since the two intensities are determined simultaneously, the measurement is independent of dye concentration, bleaching, and intensity fluctuation of the excitation source. Furthermore, analysis of the emission spectra permitted the detection of light scattering, binding effects, and chemical modification of the probe. A microspectrofluorometer was constructed to analyze low light level emission spectra from intracellular 1,4-DHPN. The instrument consists of a modified Leitz inverted microscope (E. Leitz, Inc., Rockleigh, NJ) with a Ploem illuminator adapted for broadband excitation and objective focusing capability. The emission spectra were collected by focusing the fluorescence from the cell onto the entrance slit of an imaging monochromator, which was scanned by a SIT camera interfaced with a computer. This permitted the acquisition of fluorescence emission spectra extending from 391-588 nm in approximately 33 ms. pHi measured in the cultured toad kidney epithelial cell line, A6, was 7.49 +/- 0.04 (n = 12) with an external pH of 7.6. A6 cells were found to regulate pHi in response to both acute acid and alkali loads and maintained pHi relatively constant over a wide range of external pH values. The technique described in this report overcomes several of the difficulties encountered with other fluorescent pH probes where excitation spectroscopy is required to monitor pH.     

Single cell measurement of micro-viscosity by ratio imaging of fluorescence of styrylpyridinium probe

In aqueous solution, compounds containing the styrylpyridinium group showed dual fluorescence, in which excitation at either 469 or 360 nm each produced an emission band around 600 nm. The ratio of fluorescence intensities of the two bands (R = I469/I360) was sensitive to local viscosity. The N-carboxymethyl butyl ester of DMASP was found to be able to irreversibly load into a living cell; presumably by hydrolysis involving cellular lipases it was transformed to a membrane-impermeable fluorescent carboxylate. A map of the ratio, R, from a single cell was generated using fluorescence imaging microscopy with a spectrofluorimeter in dual-excitation single-emission mode. After calibrating the ratio for the probe in water/glycerol solutions, the intracellular viscosities were obtained for a single cell of smooth muscle of a rat embryonic thoracic aorta. The intracellular viscosity is differentiated inside the cell and the obtained values 18-7 cP obey all the values reported by other laboratories. Fluorescence emission of the probe (500-650 nm) is in a very favourable region for its use with visible fluorescence microscopy, without interferences from cell or tissue auto-fluorescence. The results present ability to detect and follow small changes in the ratio of fluorescence intensities, and apparently of the micro-viscosity.     

Flow injection microscopy for the study of intracellular calcium mobilization by muscarinic agonists

The study of cellular response to chemical agonists is essential in understanding the complex functions mediated by cell surface receptors. Flow injection microscopy has been used with the CHO-M1-WT3 cell line and the fluorescent Ca2+ indicator Fura-2-AM to monitor mobilization of internal Ca2+. Repeated stimulation of cells mounted in an inverted radial flow chamber allows the direct comparison of relative intracellular Ca2+ mobilization with respect to agonist dose. The process of determining dose-response relationships is simplified since an entire dose-response curve can be constructed from a distinct set of cells. Use of flow injection lends precision to the application and removal of agonists while allowing cellular activity to be monitored throughout the stimulation and recovery processes. In this work, dose-response curves have been constructed for the muscarinic agonists carbachol, acetylcholine, and pilocarpine resulting in EC50 values of 1.7 microM, 56 nM, and 6.8 microM, respectively.     

Altered stoichiometry of the human leucocyte Na+/H+ antiport with decreasing pH.

The Na+/H+ antiport is an important regulator of cellular volume, pH and Na+ concentration in mammalian cells. The stoichiometry of this antiporter has previously been shown to be a 1:1 exchange of internal H+ for external Na+. We have investigated this stoichiometry in human leucocytes by using a novel intracellular pH-clamping technique and measuring 22Na+ influx and H+ efflux in the same cells. As internal pH was lowered, the stoichiometry of H+/Na+ exchange rose to a mean +/- S.D. of 2.23 +/- 0.69. This mechanism allows a higher H+ efflux in the face of intracellular acid stress without causing excessive intracellular Na+ overload.     

Nonequivalence of membrane voltage and ion-gradient as driving forces for the bacterial flagellar motor at low load

Many bacterial species swim using flagella. The flagellar motor couples ion flow across the cytoplasmic membrane to rotation. Ion flow is driven by both a membrane potential (V(m)) and a transmembrane concentration gradient. To investigate their relation to bacterial flagellar motor function we developed a fluorescence technique to measure V(m) in single cells, using the dye tetramethyl rhodamine methyl ester. We used a convolution model to determine the relationship between fluorescence intensity in images of cells and intracellular dye concentration, and calculated V(m) using the ratio of intracellular/extracellular dye concentration. We found V(m) = -140 +/- 14 mV in Escherichia coli at external pH 7.0 (pH(ex)), decreasing to -85 +/- 10 mV at pH(ex) 5.0. We also estimated the sodium-motive force (SMF) by combining single-cell measurements of V(m) and intracellular sodium concentration. We were able to vary the SMF between -187 +/- 15 mV and -53 +/- 15 mV by varying pH(ex) in the range 7.0-5.0 and extracellular sodium concentration in the range 1-85 mM. Rotation rates for 0.35-microm- and 1-microm-diameter beads attached to Na(+)-driven chimeric flagellar motors varied linearly with V(m). For the larger beads, the two components of the SMF were equivalent, whereas for smaller beads at a given SMF, the speed increased with sodium gradient and external sodium concentration.     

Characterization and efficacy of PKH26 as a probe to study the replication history of the human hematopoietic KG1a progenitor cell line

The PKH26 dye can, in principle, be used for the study of asymmetric cell divisions (ASDs). A requirement for the identification of ASDs based on fluorescence intensity is that the PKH26 dye is distributed equally between daughter cells at each division, but this has not been demonstrated at a single-cell level. The efficacy of PKH26 as a probe for the study of ASDs was examined using the human hematopoietic KG1a cell. An automated time-lapse fluorescent microscope system was used to determine changes in cell size and fluorescence intensity during culture, and track cell divisions. The images of daughter cells were analyzed using the Isee software to determine the distribution of PKH26 dye between daughter cells. Ratios of cell size, mean fluorescence intensity, and total fluorescence intensity were calculated by dividing the values for one daughter cell by the value of the other daughter cell. The ratios for cell size, mean intensity, and total intensity were 1.13 +/- 0.12, 1.08 +/- 0.07, and 1.15 +/- 0.14 (mean +/- SD), respectively. Thus, PKH26 is not distributed equally to both daughter cells upon cell division. However, the replication history of individual KG1a cells can be reliably deduced for up to three divisions based solely on the mean and total fluorescence intensity of the PKH26 dye, using PKH26 concentrations below the chemical and phototoxic limits (2 microM).     

Microspectrofluorometry of autofluorescence emission from human leukemic living cells under oxidative stress.

Image cytometry was applied to study the intracellular localization of autofluorescence and the influence of an oxidative stress on this emission. K562 erythroleukemia cancer cells were analyzed with a microspectrofluorometer, coupled with a Argon laser (Ar+) (363 nm). From each cell, 15 x 15 emission spectra were recorded in the 400-600 nm spectral range to generate a spectral image of autofluorescence. The intracellular locations of the autofluorescence emission and of the specific mitochondrial probe rhodamine 123 (R123) were matched. Under a 363 nm excitation, all spectra from K562 cells show equivalent profiles with a 455 nm maximum emission, near of reduced nicotinamide adenine dinucleotide-(Phosphate) solution (NAD(P)H) (465 nm maximum emission). The spatial distribution of autofluorescence is homogeneous and different from the one of R123. Hydrogen peroxide (H2O2) (200 microM) and menadione (Men) (5 microM) induce a weak spectral change and a decrease in autofluorescence intensity, down to 40% of the initial emission. Doxorubicin (Dox) induces a dose-dependent decrease in autofluorescence emission and a release of intracellular free radicals. When cells were pre-treated 1 h with 1 mM glutathione (GSH), Dox induces a lower free radicals release, no significant variation of autofluorescence intensity and a lower growth inhibitory effect. Images cytometry of autofluorescence suggest that the intracellular NAD(P)H would not be restricted to mitochondrial compartments. The release of free radicals was associated with a decrease in autofluorescence intensity, mainly attributed to NAD(P)H oxidation both inside and outside mitochondria.     

Determination of the chelatable iron pool of single intact cells by laser scanning microscopy

We have previously established a method of detecting intracellular chelatable iron in viable cells based on digital fluorescence microscopy. To quantify cellular chelatable iron, it was crucial to determine the intracellular indicator concentration. In the present study, we therefore adapted the method to confocal laser scanning microscopy, which should allow the determination of the indicator concentration on the single-cell level. The fluorescent heavy-metal indicator phen green SK (PG SK), the fluorescence of which is quenched by iron, was loaded into cultured rat hepatocytes. The hepatocellular fluorescence increased when cellular chelatable iron available to PG SK was removed from the probe by an excess of the membrane-permeable transition metal chelator 2,2'-dipyridyl (2, 2'-DPD, 5 mM). We optimized the scanning parameters for quantitatively recording changes in fluorescence and determined individual intracellular PG SK concentrations from the unquenched cellular fluorescence (after 2,2'-DPD) compared with PG SK standards in a "cytosolic" medium. An ex situ calibration method based on laser scanning microscopy was set up to determine the concentration of cellular chelatable iron from the increase of PG SK fluorescence after addition of 2,2'-DPD (5 mM). As the stoichiometry of the PG SK:Fe(2+) complex was 3:1 as long as PG SK was not limiting, cellular chelatable iron was calculated directly from absolute changes in cellular fluorescence. Using this method, we found 2.5 +/- 2.2 microM chelatable iron in hepatocytes. This method makes it possible to determine the pool of chelatable iron in single vital cells independently of cellular differences (e.g., dye loading, cell volume) in heterogeneous cell populations.