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PubMed1976

Monitoring membrane potentials in Ehrlich ascites tumor cells by means of a fluorescent dye.

1. The fluorescent intensity of the dye 3,3'-dipropylthiodicarbocyanine iodide was measured in suspensions of Ehrlich ascites tumor cells in an attempt to monitor their membrane potentials under a variety of different ionic and metabolic conditions. 2. In the presence of valinomycin, fluorescent intensity is dependent on log [K+]medium (the fluorescent intensity increased with increasing [K+]medium) where K+ replaced Na+ in the medium. Cellular K+ content also influenced fluorescent intensity in the presence of valinomycin. With lower cellular K+, fluorescent intensity in the presence of valinomycin for any given concentration was increased. 3. In the presence of gramicidin fluorescent intensity was highest in Krebs-Ringer and decreased with the substitution of choline+ for Na+. 4. The observations with ionophores are consistent with the hypothesis that the dye monitors membrane potential in these cells with an increase in fluorescence indicating membrane depolarization (internal becomes more positive). 5. The estimated membrane potentials were influenced by the way in which the cells were treated. Upon dilution of the cells from 1 in 20 to 1 in 300 the initial estimations were between -50 and -60 mV. With incubation at 1 in 300 dilution for 1 h at room temperature or a 37 degrees C, the membrane potentials ranged from -18 to -42 mV. 6. Estimations of membrane potential on the basis of chloride distribution (Cl-cell/Cl-medium) in equilibrated cells ranged from -13 to -32 mV. 7. Addition of glucose to cells equilibrated at 37 degrees C for 30 min in the presence of rotenone led to a decrease in fluorescent intensity indicating hyperpolarization. Addition of ouabain in turn led to a 70 to 100% reversal of fluorescent intensity. This hyperpolarization is therefore probably due to the electrogenic activity of the sodium pump. 8. The addition of amino acids known to require external Na+ for transport increased fluorescent intensity (depolarization) reaching a maximum at higher concentrations of amino acids. Plots of 1/deltafluorescence vs. 1/[glycine] were linear with an apparent Km of 2-3 mM. The increase in fluorescence with amino acids always required external Na+. Plots of 1/fluorescence vs. 1/[Na+]medium were also linear with an apparent Km of 29 mM. These apparent Km values compare favorably with those derived from amino acid transport studies using tracers. These data indicate that the Na+-dependent transport of amino acids in these cells is electrogenic.

Amino Acids↗

PubMed1981

Staining of blue-sensitive cones of the macaque retina by a fluorescent dye.

Intravitreal injection of a fluorescent dye, Procion yellow, results in the complete and systematic staining of a cone population in the monkey retina. These cones form an approximately regular array whose separation varies with retinal eccentricity. They are absent in the very center of the fovea, and their density peaks at 1 degree. The distribution of stained cones resembles that reported for blue-sensitive cones of other primates and, consistent with such an identification, they are found with less incidence in species having lower concentrations of blue cones.

Animals↗

PubMed1983

Staining of Plasmodium yoelii-infected mouse erythrocytes with the fluorescent dye rhodamine 123.

The cationic permeant fluorescent dye rhodamine 123 (R123) was used to stain Plasmodium yoelii-infected mouse erythrocytes. Fluorescence microscopic observations demonstrated that the parasite, but not the matrix of the infected erythrocyte, accumulated the dye. Differences in fluorescence intensity could not be found at the various developmental stages of the parasite; however, quantitation of the cell-associated dye revealed an increase in R123 uptake with parasite development. The retention of the parasite-associated dye, as measured by fluorescence microscopy and spectrophotometry after extraction of R123 with butanol, was markedly reduced by treatment of the infected erythrocytes with a proton ionophore, carbonyl cyanide m-chlorophenylhydrazone (CCCP), and an inhibitor of proton ATPase, dicyclohexylcarbodiimide (DCCD). These results indicate that the accumulation and retention of R123 in P. yoelii reflect the parasite membrane potential and suggest that the parasite plasma membrane has a membrane potential-generating proton pump.

Animals↗

PubMed2002

Forskolin-induced clearance of the fluorescent dye sulforhodamine from rat parotid intralobular duct lumen: visualization of the secretory function under a confocal laser scanning microscope.

Cyclic AMP evokes fluid secretion with bicarbonate in exocrine ducts. Clearance of fluorescent dyes from rat parotid intralobular ducts by forskolin was visualized as a fluorescence change in the duct luminal space by optical sectioning under a confocal laser scanning microscope to clarify the secretory function in the ducts. When the isolated rat parotid intralobular duct segments were superfused with membrane-impermeable fluorescent dyes during the experimental period, fluorescent dyes were passively moved into the duct space. Forskolin and isobutylmethylxanthine decreased the fluorescence of anionic dye, sulforhodamine B, and neutral dye, dextran tetramethyl-rhodamine, in the duct space, suggesting that the forskolin-induced clearance of fluorescent dyes might be the result of fluid secretion in the ducts. Methazolamide inhibited a forskolin-induced sustained decrease in duct fluorescence and intracellular acidification. Low concentrations of external Cl?, DIDS, bumetanide and amiloride did not markedly inhibit a forskolin-induced decrease in duct fluorescence. These findings suggest that a major portion of the steady decrease in duct fluorescence by forskolin was related to intracellular HCO3? production, not the uptake mechanism of external Cl?. Glibenclamide, NPPB, DPC and DMA inhibited the forskolin-induced decrease. Forskolin evokes the clearance of fluorescent dyes from duct space possibly due to fluid secretion in rat parotid ducts, associated with secretion through CFTR and DPC-sensitive anion channels of carbonic anhydrase-dependent bicarbonate linked with the Na+/H+ exchange mechanism.

1-Methyl-3-isobutylxanthine↗

PubMed2002

Preparation and flow cytometry of uniform silica-fluorescent dye microspheres.

Uniform fluorescent silica-dye microspheres have been prepared by coating preformed monodispersed silica particles with silica layers containing rhodamine 6G or acridine orange. The resulting dispersions exhibit intense fluorescent emission between 500 and 600 nm, over a broad excitation wavelength range of 460 to 550 nm, even with exceedingly small amounts of dyes incorporated into the silica particles (10-30 ppm, expressed as weight of dye relative to weight of dry particles). The fluorescent particles can be prepared in micrometer diameters suitable for analyses using flow cytometry with 488-nm laser excitation.

Journal Article↗

PubMed1999

Mechanisms of synaptic vesicle recycling illuminated by fluorescent dyes.

The recycling of synaptic vesicles in nerve terminals involves multiple steps, underlies all aspects of synaptic transmission, and is a key to understanding the basis of synaptic plasticity. The development of styryl dyes as fluorescent molecules that label recycling synaptic vesicles has revolutionized the way in which synaptic vesicle recycling can be investigated, by allowing an examination of processes in neurons that have long been inaccessible. In this review, we evaluate the major aspects of synaptic vesicle recycling that have been revealed and advanced by studies with styryl dyes, focussing upon synaptic vesicle fusion, retrieval, and trafficking. The greatest impact of styryl dyes has been to allow the routine visualization of endocytosis in central nerve terminals for the first time. This has revealed the kinetics of endocytosis, its underlying sequential steps, and its regulation by Ca2+. In studies of exocytosis, styryl dyes have helped distinguish between different modes of vesicle fusion, provided direct support for the quantal nature of exocytosis and endocytosis, and revealed how the probability of exocytosis varies enormously from one nerve terminal to another. Synaptic vesicle labelling with styryl dyes has helped our understanding of vesicle trafficking by allowing better understanding of different synaptic vesicle pools within the nerve terminal, vesicle intermixing, and vesicle clustering at release sites. Finally, the dyes are now being used in innovative ways to reveal further insights into synaptic plasticity.

Animals↗

PubMed2002

Carbachol-induced fluid movement through methazolamide-sensitive bicarbonate production in rat parotid intralobular ducts: quantitative analysis of fluorescence images using fluorescent dye sulforhodamine under a confocal laser scanning microscope.

Fluid secretion is observed at the openings of ducts in the exocrine gland. It remains unclear whether the ducts are involved in fluid secretion in the salivary glands. In the present study, we investigated the exclusion of fluorescent dye from the duct lumen by carbachol (CCh) in isolated parotid intralobular duct segments to clarify the ability of the ducts for the fluid secretion. When the membrane-impermeable fluorescent dye, sulforhodamine, was added to the superfused extracellular solution, quantitative fluorescence images of the duct lumen were obtained under the optical sectioning at the level of the duct lumen using a confocal laser scanning microscope. CCh decreased the fluorescent intensity in the duct lumen during the superfusion of the fluorescent dye, and CCh flushed out small viscous substances stained with the fluorescent dye from isolated duct lumen, suggesting that CCh might induce fluid secretion in the duct, leading to the clearance of the dye and small stained clumps from the duct lumen. CCh-induced clearance of the fluorescent dye was divided into two phases by the sensitivity to external Ca2+ and methazolamide, an inhibitor for carbonic anhydrase. The initial phase was insensitive to these, and the subsequent late phase was sensitive to these. A major portion in the late phase was inhibited by removal of bicarbonate in the superfusion solution and DPC, but not low concentration of external Cl-, bumetanide or DIDS, suggesting that methazolamide-sensitive production of HCO3-, but not the Cl- uptake mechanism, might contribute to the CCh-induced clearance of the dye from the duct lumen. These results represent the first measurements of fluid movement in isolated duct segments, and suggest that carbachol might evoke fluid secretion possibly through Ca2+-activated, DPC-sensitive anion channels with HCO3- secretion in the rat parotid intralobular ducts.

Animals↗

PubMed1988

Use of fluorescent dyes as molecular probes for the study of multidrug resistance.

Fluorescence microscopy has shown that 18 different fluorescent dyes, staining various intracellular structures in transformed hamster fibroblasts (DM-15), did not stain or stained weakly multidrug-resistant cells selected from DM-15 by colchicine. Reduced staining by fluorescent dyes was characteristic also of five other tested multidrug-resistant cell lines of hamster and mouse origin, selected by actinomycin D, colcemid, rubomycin, and ruboxyl. The intensity of staining of two revertant cell lines was similar to that of parental sensitive cells. All tested inhibitors of multidrug resistance, including weak detergent, metabolic inhibitors, calcium channel blockers, calmodulin inhibitors, and reserpine, restored normal staining of multidrug-resistant cells. The dyes accumulated in resistant cells in presence of these inhibitors left the cells several minutes after the removal of the inhibitor from the incubation medium. Sensitive cells retained the dyes for several hours. The efflux of the dyes from resistant cells is an active process since it occurred even in the presence of the dyes in the incubation medium. The efflux could be blocked by all tested inhibitors of multidrug resistance and it is possibly a basic mechanism of the reduced staining of resistant cells. These data support the idea that multidrug resistance is based on active nonspecific efflux of the drugs and indicate that the simple procedure of cell staining can be used for the detection of resistant cells and further study of the phenomenon of multidrug resistance.

Animals↗

PubMed2001

[Multidrug resistance of leukemia cells to cytostatics--the functional fluorescent dye efflux assay evaluated by flow cytometry].

The functional fluorescent dye efflux assays are used in the study of the multidrug resistance of the malignant cells to the cytotoxic drugs which is caused by the overexpression of P-glycoprotein (P-gp) or another membrane transport system--a multidrug resistance related protein (MRP). P-glycoprotein and a multidrug resistance related protein are involved in the efflux of cytotoxic drugs out of the cell and are responsible for the resistance. Fluorescent dye efflux mediated by these proteins could be evaluated by the flow cytometry. This test seems to be an optimal approach to study the multidrug resistance. With help of the specific inhibitors such as verapamil and cyclosporine A, the functional capacity of these proteins and the possibility to overcome the multiresistant phenotype can be revealed. The cell line K562 with transfected P-glycoprotein gene serves as a model system for the studying of the transport function with the use of the fluorescent substrates and P-glycoprotein inhibitors.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1966

Entry of fluorescent dyes into the sarcotubular system of the frog muscle.

1. A fluorescence microscope was used in order to detect whether fluorescent dyes can quickly diffuse into and out of certain parts of single muscle fibres of the frog.2. Several fluorescent dyes entered a system arranged at the centre of each I-band without entering the main part of the fibre.3. The dyes quickly diffused out of the fibres on washing, becoming invisible within a few minutes.4. In a hypertonic solution the fluorescent striations were more conspicuous and faded more slowly.5. It is concluded that the dyes have access to some component of the triads.6. The ;dye space' measured with Lissamine Rhodamine B200 was 1-2% of the fibre volume, and the implication of this value is discussed.7. From an analysis of the time course of entry or exit of the dye it is suggested that the principal resistance to radial diffusion of the dye is distributed along the tubules rather than at the mouths of the tubules.

Animals↗

PubMed2004

Evaluating the toxicity of Triton X-100 to protozoan, fish, and mammalian cells using fluorescent dyes as indicators of cell viability.

Three viability assays using fluorescent dyes effectively detected a loss of viability in cultures of three mammalian cell lines (H4IIE, Caco2, and HepG-2), two fish cell lines (RTgill-W1 and RTL-W1), and a ciliated protozoan, Tetrahymena thermophila, after exposure to Triton X-100, used as a model toxicant. The dyes were Alamar Blue (AB), neutral red (NR), and propidium iodide, which respectively monitored energy metabolism, lysosomal activity, and membrane integrity. A fourth fluorescent dye, 5-carboxyfluorescein diacetate acetoxymethyl ester, was problematic. For 2-h Triton X-100 exposures, mammalian cell lines were as susceptible as piscine cell lines, whereas T. thermophila was approximately twofold less sensitive as detected with AB and NR. Despite being less sensitive, cytotoxicity tests on T. thermophila could be done in spring water, which means that unlike animal cells they could be directly exposed to most industrial effluents without osmolality adjustments. Therefore, T. thermophila could be a useful complement to animal cells as alternatives to fish in toxicity testing.

Animal Testing Alternatives↗

PubMed2001

Fluorescence-quenching phenomenon by photoinduced electron transfer between a fluorescent dye and a nucleotide base.

Fluorescently labeled oligonucleotide probes have been widely used in biotechnology, and fluorescence quenching by the interaction between the dyes and a nucleobase has been pointed out. This quenching causes big problem in analytical methods, but is useful in some other cases. Therefore, it is necessary to estimate the fluorescence quenching intensity under various conditions. We focused on the redox properties of some commercially available fluorescent dyes, and investigated dye-nucleotide interactions between a free dye and a nucleotide in aqueous solution by electrochemical and spectroscopic techniques. Our results suggested that the quenching was accompanied by photoinduced electron transfer between a thermodynamically quenchable excited dye and a specific base. Several kinds of fluorescent dyes labeled to the 5'-end of oligonucleotide C10T6 were prepared, and their quenching ratios compared upon hybridization with the complementary oligonucleotide A6G10. The quenching was completely reversible and their efficiencies depended on the attached fluorophore types. The fluorescence of 5-FAM, BODIPY FL or TAMRA-modified probe was strongly quenched by hybridization.

Algorithms↗

PubMed1987

Fluorescent dye monitoring of mitochondrial changes associated with malignant cell transformation.

The fluorescent dyes, rhodamine 6G and 123, which specifically stain mitochondria, were used to examine changes in mitochondria that follow malignant transformation. The spatial distribution and shapes of mitochondria differ in untransformed and malignant-transformed cells. In untransformed C3H/10T1/2 clone 8 cells, the mitochondria were distributed radially around the nucleus, and each had a fibrous shape. In chemically transformed MCA clone 16 cells, the mitochondria were distributed randomly in the cytoplasm, and each was shaped like a short rod. Another important mitochondrial change after malignant transformation was the change in the time course of fluorescence emission from the rhodamine present in the mitochondria. A slow increase in fluorescence, which was instantaneous at the time of excitation irradiation occurred in untransformed but not in transformed cells. This slow fluorescence emission, peculiar to untransformed cells was affected by proton ionophore but not by calcium ionophore treatment. The difference in the time courses of fluorescence emission for untransformed and transformed cells may reflect differences in the quenching of the dye fluorescence. The data reported provide evidence that mitochondria are affected by malignant cell transformation.

Animals↗

PubMed1994

A novel lighting system for the measurement of dermal exposure using a fluorescent dye and an image processor.

A video technique is described which uses a fluorescent dye as a tracer to quantify dermal deposition of chemicals such as pesticides. The subject stands inside a dodecahedral arrangement of ultraviolet (u.v.) strip-lights, which cause a given area of dye to fluoresce with a consistent intensity over a range of spatial and angular positions within the lighting field. A special algorithm and an additional point source light enable local compensation factors to be calculated to allow for the reduction of apparent surface areas, due either to the foreshortening of surfaces angled to the camera or to the inverse square law for surfaces further from the camera. Measurements on planar surfaces prove that the algorithm performs well in a large central region inside the dodecahedral lighting field. Field trials of the system show that although the free-standing apparatus is large and rather unwieldy, when fastened inside a box van it is practicable, portable and self-contained. It is suitable for measuring exposure arising from spraying operations and can be used, but with more limited success, in studies of dipping operations.

Fluorescent Dyes↗

PubMed1991

Vagus nerve and spinal cord projecting neurons demonstrated by horseradish peroxidase and different fluorescent dyes.

Vagal projecting (VP) neurons were localized by intraneural injections of fluorescent dyes or cholera toxin conjugated horseradish peroxidase (CT-HRP) or by intraperitoneal injection of fluorescent dyes. Spinal projecting (SP) neurons were localized by injecting CT-HRP or contrasting dyes into the C4/C5 cord segments. No doubly labelled neurons were seen in the three nuclei known to project to both vagus nerve and spinal cord, viz., dorsal nucleus of the vagus (DNV), nucleus ambiguous complex (NAc) and the intermediate region (NI) between DNV and NAc. VP and SP neurons intermingled in the caudal parts of the NAc and DNV. In the middle part of the NAc, VP neurons congregated mostly dorsal to the SP neurons. In the rostral extremity of the NAc, SP neurons were rarely encountered. No SP neurons were seen in the rostral end of the DNV. In contradistinction to the few VP neurons in the NI, there were many SP neurons in this region. The ratios of VP to SP neurons in DNV were on the average 20 to 1 and those of VP to SP neurons in the NAc, 1.35 to 1.

Animals↗

PubMed1992

Labelling of liposomes with intercalating perylene fluorescent dyes.

The high fluorescent potential and the exceptional photostability of lipophilic derivatives of perylene-3,4:9,10-bis(dicarboximides) are utilized for the fluorescence-labelling of liposomes. The preparation of the liposomes is effected by supersonic starting from a lipid mixture consisting of the matrix lipids soy lecithin, cholesterol, alpha-tocopherol and the perylene dyes. From a multitude of perylene derivatives investigated only those are optimally incorporated into the bilayer membrane of unilamellar liposomes which are substituted at both nitrogen atoms by one or two linear hydrocarbon groups. In order to attain an optimal fluorescent quantum yield, about 200 to 300 dye molecules can be incorporated per liposome. The liposomes thus obtained have a diameter of about 70 to 80 nm, are homogeneous and may be stored for more than seven months. Neither the fluorescent properties nor the stability of these liposomes are influenced by the additional incorporation of various ara C-derivatives and lipophilic anchor groups which subsequently enable the coupling of antibodies to the liposomes. As the water-insoluble perylene dyes are incorporated into the bilayer membrane, the aqueous inner volume of the liposomes remains available for a further utilization.

Cholesterol↗

PubMed1987

Macrophages possess probenecid-inhibitable organic anion transporters that remove fluorescent dyes from the cytoplasmic matrix.

We introduced several membrane-impermeant fluorescent dyes, including Lucifer Yellow, carboxyfluorescein, and fura-2, into the cytoplasmic matrix of J774 cells and thioglycollate-elicited mouse peritoneal macrophages by ATP permeabilization of the plasma membrane and observed the subsequent fate of these dyes. The dyes did not remain within the cytoplasmic matrix; instead they were sequestered within phase-lucent cytoplasmic vacuoles and released into the extracellular medium. We used Lucifer Yellow to study these processes further. In cells incubated at 37 degrees C, 87% of Lucifer Yellow was released from the cells within 30 min after dye loading. The dye that remained within the cells at this time was predominantly within cytoplasmic vacuoles. Lucifer yellow transport was temperature dependent and occurred against a concentration gradient; therefore it appeared to be an energy-requiring process. The fluorescent dyes used in these studies are all organic anions. We therefore examined the ability of probenecid (p-[dipropylsulfamoyl]benzoic acid), which blocks organic anion transport across many epithelia, to inhibit efflux of Lucifer Yellow, and found that this drug inhibited this process in a dose-dependent and reversible manner. Efflux of Lucifer Yellow from the cells did not require Na+ co-transport or Cl- antiport; however, it was inhibited by lowering of the extracellular pH. These experiments indicate that macrophages possess probenecid-inhibitable transporters which are similar in their functional properties to organic anion transporters of epithelial cells. Such organic anion transporters have not been described previously in macrophages; they may mediate the release of naturally occurring organic anions such as prostaglandins, leukotrienes, glutathione, bilirubin, or lactate from macrophages.

Adenosine Triphosphate↗

PubMed1985

Voltage-sensitive cyanine dye fluorescence signals in lymphocytes: plasma membrane and mitochondrial components.

The origin of the cyanine dye fluorescence signal in murine and human peripheral blood leukocytes was investigated using the oxa- and indo-carbocyanines di-O-C5(3) and di-I-C5(3). Fluorescence signals from individual cells suspended with nanomolar concentrations of the dyes were measured in a flow cytometer modified to permit simultaneous four-parameter analysis (including two-color fluorescence or fluorescence polarization measurements). The contributions of mitochondrial membrane potential (psi m) and plasma membrane potential (psi pm) to the total voltage-sensitive fluorescence signal were found to depend on the equilibrium extracellular dye concentration, manipulated in these experiments by varying the ratio of dye to cell density. Hence, conditions could be chosen that amplified either the psi m or the psi pm component. Selective depolarization of lymphocytes or polymorphonuclear leukocytes (PMN) in mixed cell suspensions demonstrated that defining the partition of dye between cells and medium is requisite to assessing the heterogeneity of cell responses by cyanine dye fluorescence. At extracellular dye concentrations exceeding 5 nM in equilibrated cell suspensions, both mitochondrial and plasma membrane dye toxicity were observed. In murine splenic lymphocytes, plasma membrane toxicity (dye-induced depolarization) was selective for the B lymphocytes. Certain problems in calibration of psi pm with valinomycin at low dye concentrations and perturbations of psi pm by mitochondrial inhibitors are presented. These findings address the current controversy concerning psi m and psi pm measurement in intact cells by cyanine dye fluorescence. The finding of selective toxicity at low cyanine dye concentrations suggest that purported differences in resting psi m among cells or changes in psi pm with cell activation may reflect variable susceptibility to dye toxicity rather than intrinsic cell properties.

B-Lymphocytes↗