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PubMed2000

Effects of fluorescent dyes on selectin and integrin-mediated stages of adhesion and migration of flowing leukocytes.

Fluorescent dyes assist visualisation of leukocytes for intravital studies of adhesion or for in vitro studies utilising whole blood. We have used in vitro flow-based assays to investigate the effects of three fluorescent dyes (acridine orange, AO, 5-100 microg/ml; calcein-AM, C-AM, 5-20 microg/ml; rhodamine 6G, R6G, 10-100 microg/ml) on adhesion and migration of isolated neutrophils and mononuclear cells. AO had little effect on the number or velocity of neutrophils rolling on P-selectin presented by a surface coated with platelets. However, AO did cause a dose- and time-dependent conversion of rolling to immobilisation. Pretreatment of neutrophils with an antibody against CD18 prevented this conversion to stationary adhesion, indicating that beta(2) integrins were activated by AO. C-AM had little effect on neutrophil behaviour, but tended to cause some immobilisation at the highest concentration. R6G did not affect the number of neutrophils that bound to the platelet monolayer or the percentage rolling, but the rolling velocity of the neutrophils was increased in a dose-dependent manner. None of the dyes impaired the ability of neutrophils to respond to formyl peptide by converting from rolling to stationary adhesion. Neither C-AM nor R-6G reduced the number of flowing neutrophils or mononuclear cells binding to endothelial cells stimulated with tumour necrosis factor. Interestingly, R-6G inhibited transendothelial migration of mononuclear cells but not neutrophils, while C-AM did not affect transmigration of either cell type. The dose-dependent effects of dyes should be taken into consideration when designing any experimental protocol. AO does not appear to be a suitable dye for adhesion studies. R6G and C-AM can be used at approximately 10 microg/ml (a concentration at which cells can be clearly visualised) although R-6G specifically inhibits the migratory response of mononuclear cells.

Acridine Orange↗

PubMed1988

Effects of kainic acid lesions in lateral geniculate nucleus: activity dependence of retrograde axonal transport of fluorescent dyes.

Kainic acid lesions in the dorsal lateral geniculate nucleus of rats block the retrograde axonal transport of fluorescent dyes in corticogeniculate neurons without affecting the retrograde transport of D-aspartate or the orthograde transport of radiolabelled proteins in these neurons. This blocking of dye transport does not appear to be a consequence of kainic acid-induced damage to axon terminals in the geniculate since retinal ganglion cells are still able to transport dyes retrograde. A more likely explanation for these results is that fluorescent dye transport requires electrical activity in neurons, and elimination of the geniculate afferents to visual cortex reduces impulse traffic in cortical output fibers to a level below that required to support detectable dye transport. This interpretation is supported by the observation that kainic acid lesions also reduce retrograde transport of dyes in cortical neurons which project to the superior colliculus. Electrical stimulation in the subcortical white matter restores the transport of dye compounds in corticogeniculate neurons: evidence consistent with an activity-dependent mechanism of retrograde transport for these substances. These results provide evidence that axon terminals of retinal ganglion cells and corticogeniculate neurons survive in kainate-lesioned geniculates and are capable of normal neuronal function.

Amidines↗

PubMed1991

Photoinactivation of neurones axonally filled with the fluorescent dye 5(6)-carboxyfluorescein in the pond snail, Lymnaea stagnalis.

We describe a new, simple and reliable technique to fill molluscan neurones from their cut axons with sufficient fluorescent dye for photoinactivation experiments. The fluorescent dye 5(6)-carboxyfluorescein (5-CF) travels quickly up the nerves of the gastropod mollusc, Lymnaea stagnalis into the buccal ganglia and fills the cell bodies in 1-3 h. 5-CF filled neurones can be located in the intact ganglia with low intensity blue light. Impalement shows that they are alive and show normal resting, action and synaptic potentials. Intense laser light (wavelength 442 nm, intensity 0.5 MW.m-2) kills all the 5-CF filled cells in less than 5 min in laboratory reared snails. Unstained neurones are not killed. 5-CF fills neurones quicker than Lucifer yellow (LY) when the dye is applied axonally. Neurones stained with Lucifer yellow do not contain sufficient dye to be killed with 5 min laser illumination, but this irradiation reduces the membrane resistance to less than 25%.

Animals↗

PubMed1988

A quantitative analysis of the retrograde axonal transport of 4 different fluorescent dyes in peripheral sensory and motor neurons and lack of anterograde transport in the corticospinal system.

Many fluorescent dye compounds are transported by axons in retrograde and anterograde directions. In the present study the uptake and retrograde axonal transport of 4 chemically related fluorescent dyes was evaluated in the peripheral nervous system of adult mice. Anterograde transport was studied in the corticospinal tract of adult rats. In addition to confirming the previously reported intra-axonal transport of Rhodamine-B-isothiocyanate, we report the transport of Rhodamine-X-isothiocyanate. Sulforhodamine-101-acid chloride and Lissamine rhodamine-B-sulfonyl chloride. By using the fluorescence intensity of the labeled motor and sensory neurons as well as cell counts of fluorescently labeled motor neurons and percent of labeled dorsal root ganglia (DRG) cells, we were able to quantitate the amount of retrograde transport of a given fluorescent compound. The two dyes with isothiocyanate groups available for conjugation were transported in higher amounts compared to the dyes containing sulfonyl chloride groups. No anterograde transport in the corticospinal system was observed. We conclude that the 4 dyes described are useful for retrograde neuroanatomical tracing experiments. We describe methods for quantifying the amount of retrograde transport by peripheral motor and sensory neurons.

Animals↗

PubMedDate not supplied

[Activation of Fe2+-induced chemiluminescence in human blood low density lipoproteins by the fluorescent dye C-525].

The effect of the fluorescent dye 2,3,5,6-1H,4H-tetrahydro-9-(2'- benzoimidazolyl)-quinolizin-(9,9a,1-gh)coumarin (C-525) on the chemiluminescence (CL) flash kinetics accompanying Fe(2+)-induced free radical lipid hydroperoxide decomposition in low density lipoproteins (LDL) have been studied. C-525 was found to increase CL flash intensity by a factor of more than 2000 at the concentration of 4 mkM without any influence on the CL kinetics. The mechanism of the CL amplification is apparently energy transfer from the primary excited product of lipid peroxyl radical recombination to fluorescent level of the C-525. A concentration dependencies of CL amplification degree by C-525 on the LDL concentration were revealed to be bell-shaped, so that for 0.01 mg/ml of LDL protein the maximum amplification degree was observed at 4 mkM of C-525, whereas for 0.02 mg/ml of LDL--at 8 mkM. For the elucidation of these effects the properties of C-525 as hydrophobic fluorescent probe have been used. The concentration dependencies of C-525 fluorescence in the LDL suspension were found to be similar to that of CL amplification by C-525. These data pointed to the fact that concentration dependencies of the CL amplification by C-525 was conditioned by its accumulation in a hydrophobic phase of lipoproteins, so that the effective dye concentration was determined by both the amount of C-525 added to the suspension and the concentration of LDL.

Coumarins↗

PubMed2002

Physical modelling of solute transport in porous media: evaluation of an imaging technique using UV excited fluorescent dye.

The development and evaluation of a 2-dimensional physical model, which is designed to assist in the characterisation of complex solute transport problems in porous media, is described. The laboratory model is a transparent 2-dimensional porous media of nominal thickness and uses a non-invasive imaging technique in conjunction with a fluorescent dye tracer (sodium fluorescein) to monitor solute movements. Under ultraviolet (UV) illumination the dye emits visible light which is imaged by a CCD (Charge Coupled Device) camera. The image is processed to estimate the 2-dimensional distribution of tracer concentrations. The system can successfully model a simple contaminant plume within a homogenous porous matrix constructed from glass beads (60-100 microm). Experimental results show that transverse dispersion coefficient was 3.9 x 10(-10) m2/s when sodium fluorescein transported in porous matrix with a walter velocity of 5.71 x 10(-6) m/s. The low transverse dispersion coefficient suggests that the molecular diffusion of solute cannot be neglected under low velocity of the water. The advantages of using UV rather than an ordinary light system are a reduction in noise and experimental errors. Errors due to light dispersion within the model are shown to be negligible for the current model. Since contaminant with aromatic rings are usually fluorescent and biological samples can be labelled by fluorescent dye, this imaging technique using UV excited fluorescent dye will be used to investigate biodegradation process in porous media.

Calibration↗

PubMed1963

Adsorption and fluorescence of fat-soluble fluorescent dyes on class I and class III Saccharomyces cerevisiae.

Lycette, R. M. (Illinois Institute of Technology, Chicago) and L. R. Hedrick. Adsorption and fluorescence of fat-soluble fluorescent dyes on class I and class III Saccharomyces cerevisiae. J. Bacteriol. 85:1-6. 1963.-Class III cells of the brewery yeast Saccharomyces cerevisiae flocculate, but class I cells do not, when cultured in an organic YM medium. Class III cells adsorb the fat-soluble stilbyl-triazole fluorescent dye (STC) more readily and produce more fluorescence than do class I cells. This suggests that cells of the class III yeasts have more lipids or more phospholipids on their surface than do cells of class I yeasts. Fluorescence of STC in the absence of cells is pH-independent, but adsorption of the compound on the surface of cells, with the associated fluorescence, is pH-dependent. As the pH level was decreased within the range of pH 9 to 0, and as the temperature was elevated within the range of 0 to 40 C, there was an increase in adsorption and a corresponding increase in fluorescence. Maximal adsorption was attained in less than 15 min when the cells and dye were shaken at 26 C. Cells of class III adsorbed 2.5 mug of STC per 3.25 mg (dry wt) of cells. The kinetics of adsorption follow the Langmuir monomolecular model.

Adsorption↗

PubMed1999

Leakage responses to L-NAME differ with the fluorescent dye used to label albumin.

Nitric oxide synthase (NOS) inhibitors have been reported to increase as well as to decrease microvascular transport of macromolecules in a variety of models. This study was performed to determine whether the influence of NOS inhibition on albumin leakage was dependent on the fluorescent dyes used to label albumin. Albumin leakage was assessed in rat mesenteric venules during control conditions and after exposure to the NOS inhibitor NG-nitro-L-arginine methyl ester (L-NAME). Albumin was labeled with any one of four dyes: FITC, sulforhodamine 101 [Texas Red (TR)], dichlorotriazinyl aminofluorescein (DTAF), or Oregon Green 514 (OG). Superfusion with L-NAME (10(-4) M) was accompanied by an increase in leakage of FITC-labeled albumin (n = 12) but not of albumin labeled with DTAF (n = 10), TR (n = 10), or OG (n = 4). In vessels perfused with both FITC- and TR-labeled albumin (n = 12), superfusion with L-NAME increased leakage of FITC- but not TR-labeled albumin. In conclusion, albumin leakage responses to L-NAME differ among various fluorescent dyes. Therefore, caution is advised in comparison of albumin leakage results that utilize different fluorescent dyes.

Animals↗

PubMed1998

The interactions between the fluorescent dye thiazole orange and DNA.

The interaction of the fluorescent dye thiazole orange (TO) with nucleic acids is characterized. It is found that TO binds with highest affinity to double-stranded (ds) DNA [log (K) approximately 5.5 at 100 mM salt], about 5-10 times weaker to single-stranded polypurines, and further 10-1000 times weaker to single-stranded polypyrimidines. TO binds as a monomer to dsDNAs and poly(dA), both as a monomer and as a dimer to poly(dG) and mainly as a dimer to poly(dC) and poly(dT). The fluorescence quantum yield of TO free in solution is about 2 x 10(-4), and it increases to about 0.1 when bound to dsDNA or to poly(dA), and to about 0.4 when bound to poly(dG). Estimated quantum yields of TO bound to poly(dC) and poly(dT) are about 0.06 and 0.01, respectively. The quantum yield of bound TO depends on temperature and decreases about threefold between 5 and 50 degrees C.

Benzothiazoles↗

PubMed2000

Visualization of phospholipid domains in Escherichia coli by using the cardiolipin-specific fluorescent dye 10-N-nonyl acridine orange.

Cardiolipin (CL)-specific fluorescent dye 10-N-nonyl-acridine orange (NAO) was used to visualize CL distribution in Escherichia coli cells of different phospholipid compositions. In a filamentous mutant containing only anionic phospholipids, green fluorescent spots were observed along the filaments at approximately regular intervals. Three-dimensional image reconstruction obtained by optical sectioning and a deconvolution algorithm revealed NAO-binding domains in the plane of the cell membrane. Substantial red fluorescence emission of bound NAO supported labeling of CL-containing domains. These structures were not found in mutants deficient in CL biosynthesis. The domains were also observed mostly in the septal region and on the poles in cells of normal size with wild-type phospholipid composition.

Acridine Orange↗

PubMed2005

Optical glucose detection across the visible spectrum using anionic fluorescent dyes and a viologen quencher in a two-component saccharide sensing system.

A very general system is described in which anionic fluorescent dyes possessing a wide range of absorbance and emission wavelengths are used in combination with a boronic acid-modified viologen quencher to sense glucose at pH 7.4 in buffered aqueous solution. The present study demonstrates this capability with the use of eleven anionic fluorescent dyes of various structural types. Signal modulation occurs as the monosaccharide binds to the viologen quencher and alters its efficiency in quenching the fluorescence of the anionic dyes. The degree of quenching and the magnitude of the glucose signal were found to correlate roughly with the number of anionic groups on the dye. Optimal quencher : dye ratios were determined for each dye to provide a fairly linear signal in response to changes in glucose concentration across the physiological range.

Anions↗

PubMed2004

Methods to maximise the staining of fungal propagules with fluorescent dyes.

The spores and conidia of most fungi have very thick and resistant cell walls that severely impede the staining with fluorescent dyes to allow epifluorescence microscopy to be employed for their direct detection and quantification in natural habitats. In this study, oxidation by sodium hypochlorite and microwave irradiation (MWI) were used to enhance the staining of Aspergillus fumigatus and Penicillium brevicompactum conidia with six fluorescent dyes. Sodium hypochlorite resulted in high percentages of stained conidia (up to 98.8% with 4',6-diamidino-2-phenylindole [DAPI]), but had to be removed prior to staining with consequent heavy conidia losses. By contrast, MWI gave very high percentages, while its enhancement of fluorescence intensity facilitated observation by epifluorescence microscopy.

Aspergillus fumigatus↗

PubMed1994

Antibody-mediated fluorescence enhancement based on shifting the intramolecular dimer<-->monomer equilibrium of fluorescent dyes.

A novel concept is described for directly coupling fluorescence emission to protein-ligand binding. It is based on shifting the intramolecular monomer<-->dimer equilibrium of two fluorescent dyes linked by a short spacer. A 13-residue peptide, recognized by a monoclonal antibody against human chorionic gonadotrophin (hCG), was labeled with fluorescein (F) and tetramethylrhodamine (T) at its N- and C-terminus, respectively. Spectral evidence suggests that when the conjugate is free in solution, F and T exist as an intramolecular dimer. Fluorescence quenching of fluorescein and rhodamine is approximately 98% and approximately 90%, respectively, due to dimerization. When the double-labeled peptide is bound to anti-hCG, however, the rhodamine fluorescence increases by up to 7.8-fold, depending upon the excitation wavelength. This is attributed to the dissociation of intramolecular dimers brought about by conformational changes of the conjugate upon binding. Fluorescein fluorescence, on the other hand, was still quenched because of excited-state energy transfer and residual ground-state interactions. Antibody binding also resulted in a approximately 3.4-fold increase in fluorescence anisotropy of the peptide. These changes in intensity and anisotropy allow direct measurement of antigen-antibody binding with a fluorescence plate reader or a polarization analyzer, without the need for separation steps and labeling antibodies. Because recent advances in peptide technology have allowed rapid and economical identification of antigen-mimicking peptides, the double-labeled peptide approach offers many opportunities for developing new diagnostic assays and screening new therapeutic drugs. It also has many potential applications to techniques involving recombinant antibodies, biosensors, cell sorting, and DNA probes.

Amino Acid Sequence↗

PubMed1991

Quantitative analysis of polymerase chain reaction (PCR) products using primers labeled with biotin and a fluorescent dye.

PCR primers covalently labeled with biotin and a fluorescent dye allow immobilization and separation of the products which can be quantitatively analyzed subsequently. The procedure we have developed circumvents electrophoretic separation and radioactive labeling. Exact quantitative analysis of reaction products is feasible during the logarithmic phase of amplification when Taq polymerase is not limiting, as it is during the plateau phase of the reaction. With appropriate standardization the procedure can be used for routine diagnostic purposes.

Base Sequence↗

PubMed2005

[Study of achieving white organic LED by fluorescence dye].

Some hybrid organic LEDs are made by using fluorescence dye and InGaN blue-light chip and the possibility of achieving white organic LED is investigated according to light conversion theory. Firstly, the LEDs made by normal method and double-dotting glue method are studied. It is found that the double-dotting glue method is too complex and the LEDs made by this way is low-luminance, so it isn't fit to make LED. Secondly the different weight ratio of 1/1/100, 1/1/200, 2/1.5/100 ax-17/zq-13/AB LEDs are manufactured. The 2/1.5/100 LED's color coordinate is (0.32, 0.30), approaching to white point and the correlative color temperature is 6290K which is close to sunlight. So it is a relatively ideal white lamp-house.

Color↗

PubMedDate not supplied

Detection of human leukemia cells with multidrug-resistance phenotype using multilabeling with fluorescent dyes.

Reduced accumulation of multiple drugs is a characteristic of cells overexpressing P-glycoprotein. This phenotype is referred to as multidrug-resistance (MDR). A protocol based on reduced accumulation of fluorescent dyes is proposed for discriminating MDR cells in cell populations. The combination of three fluorescent dyes, Hoechst 33342, rhodamine 123 and Nile red, with different intracellular targets, has been designed to characterize cells with different levels of resistance, using image cytometry. The fluorescence intensity of each dye was quantified in living cells. The protocol was applied to human leukemia cell lines, (K562, K562/ADR, CCRF-CEM, CEM/VLB100, CEM/VM-1). The effect of verapamil on dye accumulation is emphasized.

Antineoplastic Agents↗

PubMed2004

Use of the fluorescent dye 10-N-nonyl acridine orange in quantitative and location assays of cardiolipin: a study on different experimental models.

The fluorescent dye 10-N-nonyl acridine orange (NAO) is extensively used for location and quantitative assays of cardiolipin in living cells on the assumption of its high specificity for cardiolipin; however, the limits and the mechanism of this specificity are not clear. Moreover, whether factors such as the membrane potential in mitochondria may limit the consistency of the results obtained by this method is open to discussion. The aim of this research was to investigate the effects of some experimental factors on the selective fluorescence of NAO in the presence of cardiolipin in artificial and natural membranes (mitochondria). The results show that the fluorescence of NAO, due to interaction with cardiolipin, is significantly modified by factors that control the spatial arrangement of cardiolipin molecules within the space of the membrane under investigation. Moreover, the present observations suggest that the specific effect of cardiolipin is to facilitate the dimerization of this fluorescent dye, thus confirming that reliable measurements of cardiolipin concentration can be obtained only when the NAO/cardiolipin molar ratio is equal to 2. The finding is also reported that in isolated respiring mitochondria the interaction of NAO with cardiolipin is somewhat related to the respiratory state of mitochondria.

Aminoacridines↗

PubMed1993

An improved method for studying microvascular geometry using fluorescent dyes: preventing dye extravasation, preserving dye integrity and enhancing tissue morphometry.

A procedure for stabilizing fluorescent markers used to study the microvascular geometry and morphometry of muscle tissue is described. The procedure involves fluorescent labeling of plasma, fixation of muscle tissue in 10% buffered formalin, and quick freezing. This procedure prevents extravasation of the fluorescent dyes out of the capillaries as frequently seen in other muscle microvascular techniques, thereby greatly increasing the time that capillaries are visible. We found that formalin may actually increase the rate of fluorochrome bleaching by photo-oxidation, but the increased rate of bleaching is more than offset by the greater concentration of dye trapped in the capillaries. Further, formalin fixation results in little distortion of the muscle fibers themselves, making this approach ideal for morphometric studies.

Animals↗