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At least 109 records · Page 6Linked to original sources
PubMed1999

Improved DNA sequencing accuracy and detection of heterozygous alleles using manganese citrate and different fluorescent dye terminators.

The use of dideoxynucleotide triphosphates labeled with different fluorescent dyes (dye terminators) is the most versatile method for automated DNA sequencing. However, variation in peak heights reduces base-calling accuracy and limits heterozygous allele detection, favoring use of dye-labeled primers for this purpose. We have discovered that the addition of a manganese salt to the PE Applied Biosystems dye-terminator sequencing kits overcomes these limitations for the older rhodamine dyes as well as the more recent dichloro-rhodamine dyes (dRhodamine and BigDyes). Addition of manganese to reactions containing dRhodamine-based dye terminators produced the highest base-calling accuracy. This combination resulted in the most uniform electropherogram profiles, superior to those produced by BigDye terminators and published for dye primers, and facilitated detection of heterozygous alleles.

Alleles↗
PubMed1985

Visualization of the mitochondria of Toxoplasma gondii-infected mouse fibroblasts by the cationic permeant fluorescent dye rhodamine 123.

The mitochondria of living mouse fibroblasts infected with Toxoplasma gondii were monitored with the cationic permeant fluorescent dye rhodamine 123. Fluorescence microscopy revealed that host cell mitochondria accumulated at the cytoplasmic surface of parasitophorous vacuoles and increased the dye uptake in the periparasitophorous vacuole as T. gondii multiplied.

Animals↗
PubMed1994

Visualization of human choroidal ganglion cells with the supravital fluorescent dye 4-(4-diethylaminostyryl)-N-methylpyridium iodide.

The distribution and morphology of ganglion cell bodies and nerve fibers in supravital human choroidea is studied by staining with the fluorescent dye 4-(4-diethylaminostyryl)-N-methylpyridium iodide (4-Di-2-ASP). We have used the choroids from human donor eyes donated for cornea transplantation. The isolated tissues were rinsed in ringer solution and processed within up to 24 h after death with 3 microM of 4-Di-2-ASP in Ringer for 5-60 min at different temperatures (4-37 degrees C). After incubation, choroid whole flat mounts were observed in a fluorescence microscope. A delicate network of nerve fibers could be distinguished as soon as 5 min after immersion in 4-Di-2-ASP. In addition, labelled ganglion cell perikarya could be seen after longer incubation periods. They occurred individually or grouped in small ganglia of up to 8 cells and were linked together by various processes, which could also be traced to blood vessels. The fluorescent dye 4-Di-2-ASP provides a quick and simple method to visualize the innervation of the human choroid, including local ganglion cells. This nontoxic fluorescent technique will permit further manipulation and electrophysiological investigation of choroidal innervation.

Adolescent↗
PubMed1982

Anterograde and transcellular transport of a fluorescent dye, bisbenzimide, in the rat visual system.

Fluorescent labeling was seen in rat brain after injections of 10 microliters of 10% bisbenzimide into the posterior chamber of the eye. Small and spindle shaped fluorescent nuclei were seen in the ipsilateral optic nerve, chiasm and contralateral optic tract, while larger apparently neuronal nuclei were seen in regions such as the lateral geniculate and superior colliculus where the retinal projection terminates. These experiments suggest that, like radioactive compounds, fluorescent dyes undergo anterograde and transcellular transport following large injections. Although limited by sensitivity and the large injection volumes required, this anterograde transport and transcellular transfer of bisbenzimide may be useful in identifying cell populations near the terminal projections of specific neuronal pathways.

Afferent Pathways↗
PubMedDate not supplied

Evanescent field excitation and measurement of dye fluorescence in a metallic probe near-field scanning optical microscope.

We introduce a method of dye fluorescence excitation and measurement that utilizes a near-field scanning optical microscope (NSOM). This NSOM uses an apertureless metallic probe, and an optical system that contains a high numerical aperture (NA) objective lens (NA= 1.4). When the area which satisfies NA < 1 is masked, the objective lens allows for the rejection of possible transmitted light (NA < 1) through the sample. In such conditions, the focused spot consists of only the evanescent field. We found that this NSOM system strongly reduces the background of the dye fluorescence and allows for the measurement of the fluorescence intensity below the diffraction limit of the excitation source.

Journal Article↗
PubMed2000

Migration of phagocytotic cells and development of the murine intraretinal microglial network: an in vivo study using fluorescent dyes.

This work was undertaken to study whether retinal ganglion cell (RGC) death, which occurs during postnatal development of the mouse retina could aid in assessing the topological and chronological pattern of microglial cell migration. The study was conducted from postnatal day 0 (P0) to adulthood. The fluorescent dyes Fluorogold (FG) or (4-[4-didecylaminostyryl]-N-methylpyridinium iodide (4Di-10ASP) used in this study, were transported retrogradely to the RGC soma when either dye was injected into the superior colliculus (SC) at P0. Some of these labeled RGCs die due to natural apoptosis during this stage of development and are phagocytosed by microglial cells, which move to the site of RGC death, to become labeled with the same dye. The retinas were examined to quantify the microglial cells from P5 to adulthood. In addition, the reaction of microglia to optic nerve crush was studied in adult animals. Both dyes labeled RGCs in the contralateral retina and a few RGCs in the retina ipsilateral to the injected SC. The density of labeled RGCs decreased by 22% between P5 and P7. During this phase, microglial cells become visible as they ingested the fluorescent detritus of the dying RGCs. Microglial cells were evenly distributed across the entire retinal surface and migrated to the outer plexiform layer. Migrating microglia consecutively altered their morphology from the amoeboid to the ramified form. In terms of intracellular storage of the dyes, resident microglial cells retained the fluorescent dye 4Di-10ASP over a period of 12 months. In contrast, FG was completely transferred from the RGCs and microglial cells to intramural cells (pericytes) of the retinal capillaries after 10 months. This resulted in delineation of the entire intraretinal vascular network. Finally, resident retinal microglial cells were also activated by injury to the adult optic nerve and phagocytosed degenerating neurons. Retinal microglial cells can be monitored with vital fluorescent dyes while they migrate across the retina and establish their intra-retinal network. It is possible to label microglia with lipophilic dyes and they remain labeled for a long time. In addition, intramural pericytes can be labeled by slow release of FG from RGCs and microglial cells. The findings suggest that ingested fluorescent dyes having different properties can be used to study different populations of retinal cells in vivo.

Age Factors↗
PubMed2000

Visualization of the secretory process involved in Ca2+-activated fluid secretion from rat submandibular glands using the fluorescent dye, calcein.

The central feature of fluid and electrolyte secretion by salivary acinar cells is transepithelial Cl- movement as a driving force for the secretion. However, little is known about the membrane localization and regulation by agonists of various anion channels. To characterize the anion transport and fluid secretion, we visualized the secretory process induced by the cholinergic agonist, carbachol (CCh), using the anionic fluorescent dye, calcein, under a confocal laser scanning microscope. The fluorescence of calcein loaded into the isolated acini was spread diffusely throughout the cytoplasm and was less intense in the secretory vesicles which occupied the apical pole. Cytoplasmic calcein was released into intercellular canaliculi just after the addition of CCh, depending upon a rise in [Ca2+]i by Ca2+ release from intracellular stores. Thereafter, the formation of watery vacuoles connected with intercellular canaliculi was visualized in the calcein-loaded acini, depending upon external Ca2+. Both the calcein release and vacuole formation were inhibited by suppressing the Ca(2+)-activated K+ efflux. The calcein release was also affected by the external anion substitution, suggesting that calcein is released through an anion channel. In the isolated, perfused glands, CCh-induced fluid secretion was sustained in two phases, whereas the loaded calcein was initially and transiently released into the saliva. By revealing the [Ca2+]i dependence and sensitivities to channel blockers, our results suggest that the initial phase of CCh-induced fluid secretion was evoked in association with the release of the organic anion, calcein, and the late phase of fluid secretion, during which calcein is less permeable, was associated with the formation of watery vacuoles. Thus, the anion channels possessing the distinct property of anion permeation may be activated in the initial phase and late phase. These results indicate that the anionic fluorescent dye, calcein, is useful for visualizing the process of Ca(2+)-dependent fluid secretion, and for clarifying the relation between fluid secretion and anion transport.

Animals↗
PubMed1980

Synthesis and study of conformationally defined enantiomers of local anesthetics and conformationally defined enantiomers of fluorescent dyes designed to label electrically excitable membranes.

Conformationally defined enantiomeric local anesthetics and fluorescent dyes were synthesized. Neither the local anesthetics nor the fluorescent probes showed stereospecificity in interacting with nerve membranes. The fluorescence signals generated by the dyes showed excellent correlation with the time course and shape of the nerve action potential.

Action Potentials↗
PubMed1980

Chemotactic factor-induced membrane potential changes in rabbit neutrophils monitored by the fluorescent dye 3,3'-dipropylthiadicarbocyanine iodide.

Rabbit neutrophil leucocytes take up the cationic, fluorescent dye 3,3'-dipropylthiadicarbocyanine iodide (DiS-C3-(5)). Treatment with valinomycin and K+ then produces characteristic changes in suspension fluorescence that indicate that the dye enters the cells in a potential-dependent fashion and that the resting membrane potential lies between -66 and -86 mV. The peptide, N-fMet-Leu-Phe, a potent chemoattractant for neutrophils, added to stained cell suspensions, induces fluorescence intensity changes. These occur over an 8-10 min period. The time course of this response is profoundly affected by the omission of Ca2+ from the medium. When this ion is present (1.26 mM) a small, transient increase in intensity is observed, superimposed on a sustained decrease. On the other hand, in the absence of added Ca2+ a large, transient increase is observed. The ED50 for this is 1.1 x 10(-10) M. These changes are not elicited by N-fMet-Phe (10(-9) M) and are inhibited by the antagonist Boc-Leu-Phe-Leu-Phe. However, a component of zymosan-activated rabbit plasma, which is complement-derived, induces identical fluorescence changes that are not inhibited by the antagonist, confirming that neutrophil activation by complement operates through an independent receptor. The fluorescence responses to the chemotactic peptide and the activated-plasma component may be interpreted in terms of changes in neutrophil membrane potential brought about by alterations in cell ionic permeability at an early stage during activation. The transient increase corresponds to a depolarisation that may be associated with a change in Na+ permeability, while the sustained decrease corresponds to a membrane hyperpolarisation.

Anaphylatoxins↗
PubMed1975

Fluorescent dye method for determination of the bilirubin-binding capacity of serum albumin.

A simple, rapid microfluorometric method utilizing a fluorescent dye, Direct Yellow 7, for quantitative measurement of serum albumin-binding capacity for bilirubin has been developed. The dye binds exclusively to serum albumin with enhancement of fluorescence. The dye shares bilirubin-binding sites on albumin with up to 2 mols of bilirubin per mol of albumin with differing affinities to the first and second sites. Lowered pH reduces albumin-binding capacity and exposure to light increases binding capacity of icteric sera. The binding capacity of adult human sera is greater than that of human cord sera or purified human serum albumin compared on albumin molar basis.

Bilirubin↗
PubMed2000

Penetration pathways of fluorescent dyes in human hair fibres investigated by scanning near-field optical microscopy.

Thin cross-sections of human hairs were investigated by scanning near-field optical microscopy (SNOM) and confocal laser scanning microscopy (CLSM) after penetration of a fluorescent dye. The same samples were measured with both techniques to compare the observed structures. The images obtained from the two methods show nearly identical structures representing pathways of the dye molecules in hairs. The SNOM images provide a higher resolution than the CLSM images. Therefore, SNOM is believed to be a suitable method for investigations at a resolution of 100 nm on penetration pathways of fluorescent dyes such as the cell membrane complex pathway in cross-sections of hairs.

Fluorescent Dyes↗
PubMed1985

Lymphocyte membrane potential and Ca2+-sensitive potassium channels described by oxonol dye fluorescence measurements.

A method is described for quantitative measurement of lymphocyte transmembrane electrical potential difference (psi) by flow cytometric recording of the oxonol dye fluorescence of single cells. Both the simultaneous collection and analysis of multiple optical parameters and the use of a negatively charged oxonol probe allowed more accurate measurement of psi than may be obtained by bulk cell suspension techniques employing cationic voltage indicators. Mouse spleen and human blood lymphocyte psi was calculated to be -70 mV. T and B lymphocytes maintain a constant psi as extracellular K+ is varied from 2 to 10 mM and the deviation from K+ equilibrium potentials (EK) is shown to result from Na+ permeability. At [K+]o values greater than 10 mM, lymphocytes behave as K+ electrodes. Examination of lymphocyte subsets showed that hyperpolarization induced by the Ca2+ ionophore A23187 occurs only in T cells. This response was identified as activation of a Ca2+-sensitive K+ channel by pharmacologic manipulations. Hence, T cells depolarized by 4-aminopyridine (4-AP, 10 mM) were observed to return to resting psi by A23187-induced elevation of [Ca2+]i. Cells depolarized by quinine (100 microM) were unaffected by A23187. The Ca2+-activated channel does not contribute to resting psi in T cells since it may be selectively blocked by quinine (20 microM) or modulated by calmodulin antagonists (5 microM trifluperazine) without affecting resting psi.

4-Aminopyridine↗
PubMed1995

Methylmercury induces Ca(2+)-dependent hyperpolarization of mouse thymocytes: a flow cytometric study using fluorescent dyes.

The effect of methylmercury on mouse thymocytes was examined using fluorescent dyes for membrane potential and intracellular Ca2+. Methylmercury at concentrations of 1 microM or higher (up to 30 microM) produced hyperpolarization in a dose-dependent fashion. Charybdotoxin and quinine, but not 4-aminopyridine and tetraethylammonium, greatly suppressed methylmercury-induced hyperpolarization. Removal of external Ca2+ reduced the degree of hyperpolarization. Pretreatment of thymocytes with A23187 under Ca(2+)-free conditions abolished the hyperpolarization induced by methylmercury. Under both normal and Ca(2+)-free conditions methylmercury increased the intracellular concentration of Ca2+. The results suggest that the increase in intracellular Ca2+ is mediated through a Ca2+ release from intracellular stores as well as through influx of external Ca2+. Therefore, it is likely that methylmercury increases the intracellular concentration of Ca2+, resulting in activation of Ca(2+)-dependent K+ conductance of mouse thymocytes.

Animals↗
PubMed2006

Assessment of gamete quality for the eastern oyster (Crassostrea virginica) by use of fluorescent dyes.

Evaluation of sperm motility is the single most widely used parameter to determine semen quality in mammals and aquatic species. While a good indicator for fresh sperm viability, post-thaw motility is not always effective at predicting fertilizing ability. Techniques using fluorescent dyes can assess functionality of mammalian sperm, but have not been widely applied in aquatic organisms. The eastern oyster Crassostrea virginica is an important mollusk in the United States, and cryopreservation protocols have been developed to preserve sperm and larvae to assist research and hatchery production. In this study, protocols were developed to assess sperm cell membrane integrity and mitochondrial function by flow cytometry and to assess viability of eggs by fluorescence microscopy. The fluorescent dyes SYBR 14 and propidium iodide (PI) (to assess membrane integrity) and rhodamine 123 (R123) (to assess mitochondrial membrane potential) were used to evaluate the quality of thawed oyster sperm previously cryopreserved with different cryoprotectant and thawing treatments. Membrane integrity results were correlated with motility of thawed sperm and mitochondrial membrane potential with fertilizing ability. Fluorescein diacetate (FDA) was used to assess cytotoxicity of cryoprotectant solutions and post-thaw damage to oyster eggs. The results indicated that membrane integrity (P=0.004) and thawing treatments (P=0.04), and mitochondrial membrane potential (P=0.0015) were correlated with motility. Fertilizing ability was correlated with cryoprotectant treatments (P=0.0258) and with mitochondrial membrane potential (P=0.001). The dye FDA was useful in indicating structural integrity of fresh and thawed eggs. Exposure of eggs, without freezing, to dimethyl sulfoxide yielded higher percentages of stained eggs and fertilization rate than did exposure to propylene glycol (P=0.002). Thawed eggs were not stained with FDA (<1%) and larvae were not produced.

Animals↗
PubMed1990

Cell size specific binding of the fluorescent dye calcofluor to budding yeast.

The yeast Saccharomyces cerevisiae cell surface outside of the bud scars displayed an increasing fluorescence intensity with increasing cell size (volume), where fluorescence was due to irreversible binding of the fluorescent dye calcofluor. The increase in fluorescence intensity appeared to be due to an increase in the density of fluorescence per unit surface area of the cell. Exposure time measurements from a photomicroscope were used to quantitate fluorescence intensity on individual cells. The cell size dependent increase in fluorescence intensity was displayed by unbudded cells from stationary phase populations, and unbudded and parent cells from exponentially growing populations. Abnormally large cells generated during the arrest of cell division with alpha-factor or restrictive temperature for cdc3, 8, 13, 24, and 28 cell division cycle mutants, displayed significantly greater fluorescence intensity compared to the smaller cells generated during the arrest of division for cdc25, 33, and 35 mutant strains. Fluorescence intensity on newly emerging buds was broadly dependent on both the size of the bud, and the size of the parent cells on which the buds were growing.

Benzenesulfonates↗
PubMed1993

Mechanism of uptake of the fluorescent dye 2-(4-dimethylaminostyryl)-1-ethylpyridinium cation (DMP+) by phospholipid vesicles.

The fluorescent dye 2-(4-dimethylaminostyryl)-1-ethylpyridinium cation (DMP+) is taken up by liposomes of egg phosphatidylcholine in response to the imposition of a transmembrane potential. Entry of DMP+ into the bilayer driven by the transmembrane potential is accompanied by a change in the fluorescence emission maximum of the dye. This change reflects the movement of the dye molecules from the headgroup region of the bilayer into the region of the fatty acyl chains. It is released into the external aqueous phase on discharge of the transmembrane potential. Partition of the dye into the phospholipid bilayer is favoured by the presence of negatively charged lipids, such as dioleoylphosphatidic acid and dicetyl phosphate, in the bilayer. Stearylamine opposes entry of the dye into the bilayer. Tetraphenylboron (TPB-) increases the partitioning of DMP+ into the phospholipid bilayer even in the absence of a transmembrane potential. The fluorescence emission maximum of DMP+ under these conditions is similar to that observed in the absence of TPB- following imposition of the transmembrane potential. It is suggested that TPB- facilitates the entry of DMP+ into the fatty acyl chain regions of the phospholipid bilayer.

Drug Interactions↗
PubMed1993

Simultaneous measurement of two cations with the fluorescent dye indo-1.

The fluorescent chelator Indo-1 can simultaneously provide information about two ions, such as Ca2+ and Ba2+, if spectra after binding can be distinguished from each other and from the ion-free dye. In the case of extracellular Ca2+ and Ba2+, the three spectra were found to be unique and independent, in the sense that no one of them could be approximated by any combination of the other two. Upon binding Ba2+, Indo-1 experienced a peak shift from 470 to 455 nm, whereas the peak for Ca(2+)-bound Indo-1 occurred at 405 nm. The computer could distinguish a contribution from Ba2-bound Indo-1 just as well when it was in a mixture containing contributions from free dye and Ca(2+)-bound Indo-1. The program used all the spectral data in a rapid noniterative calculation. It found the unique combination of Ca(2+)-bound, Ba(2+)-bound, and metal-free Indo-1 spectra which best matched the data. The spectral weighting factors which formed the best combination spectrum then corresponded to the relative concentrations of the dye in the three states. From these and the dissociation constants for Ca2+ and Ba2+, it was possible to calculate both [Ca2+] and [Ba2+] from a single data spectrum. The method was also applied to Mn2+, a metal which does not generate a unique spectrum, but which quenches Indo-1.(ABSTRACT TRUNCATED AT 250 WORDS)

Barium↗