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At least 1,261 records · Page 70Linked to original sources
PubMed1982

Fluorescein appearance time curves. Continuous recordings of iris and anterior chamber dye flow contours.

Fluorescein appearance time (FAT) curves were recorded at 5-s intervals from the feline iris and anterior chamber using a modified video-frame store unit. Anterior chamber washout slopes were also recorded following intracameral injection. The iris FAT interval following intravenous injection was maximally 5 s. Passage of the dye bolus then appeared during the next 10 s, followed by a rapid (100-s) rise to maximum levels. In contrast to the iris, anterior chamber FAT curves recorded from the pupillary space showed an approximate two-minute delay after injection (mean = 1.92 +/- 0.360 minutes), followed by a gradual (20-minute) rise to maximum recordable levels. The mean anterior chamber washout rate following intracameral injection of 10(-6) g/mL of dye levels was 2.323% +/- 0.810%/min. There are potential applications of the method and a need for shorter recording intervals.

Animals↗
PubMed1988

Rapid visualization of Acanthamoeba using fluorescein-conjugated lectins.

We investigated the efficacy of fluorescein-conjugated lectins (FCLs) for the rapid visualization of Acanthamoeba species. Cultures of Acanthamoeba castellani, Acanthamoeba culbertsoni, and Acanthamoeba polyphaga were established on nonnutrient agar plates supplemented with Escherichia coli. Maximal trophozoite populations were established four to five days after initial subculturing; mature cysts were routinely noted three to six days later. At various time points, trophozoites and/or cysts were harvested and suspended in Page's saline. Following a previously described protocol, samples of the suspensions were placed on glass slides and incubated with one of a panel of 14 FCLs. At the end of the incubation period, the slides were examined using an epifluorescence microscope. Concanavalin A brightly stained both cysts and trophozoites. Wheat germ agglutinin brightly stained cysts but only minimally stained trophozoites. These results suggest that FCLs may be useful for rapid visualization of Acanthamoeba organisms in corneal infections.

Acanthamoeba↗
PubMed1997

Bactericidal antibody recognition of a PorA epitope of Neisseria meningitidis: crystal structure of a Fab fragment in complex with a fluorescein-conjugated peptide.

Class 1 outer membrane protein PorA of Neisseria meningitidis is a vaccine candidate against bacterial meningitis. Antibodies against PorA are able to induce complement-mediated bacterial killing and thereby play an important role in protection against meningococcal disease. Bactericidal antibodies are all directed against variable regions VR1 and VR2 of the PorA sequence, corresponding to loops 1 and 4 of a two-dimensional topology model of the porin with eight extracellular loops. We have determined the crystal structure to 2.6 A resolution of the Fab fragment of bactericidal antibody MN12H2 against meningococcal PorA in complex with a linear fluorescein-conjugated peptide TKDTNNNL derived from the VR2 sequence of sero-subtype P1.7,16 (residues 180-187) from meningococcal strain H44/76. The peptide folds deeply into the binding cavity of the Fab molecule in a type I beta-turn, with the minimal P1.16 epitope DTNNN virtually completely buried. The structure reveals H-bonds and van der Waals interactions with all minimal epitope residues and one essential salt bridge between Asp-182 of the peptide and His-31 of the MN12H2 light chain. The key components of the recognition of PorA epitope P1.16 by bactericidal antibody MN12H2 correspond well with available thermodynamic data from binding studies. Furthermore, they indicate the structural basis of an increased endemic incidence of infection by group B meningococci in England and Wales since 1981 associated with the occurrence of an Neisseria meningitidis escape mutant (strain-MC58). The observed three-dimensional conformation of the peptide provides a rationale for the development of a synthetic peptide vaccine against meningococcal disease.

Amino Acid Sequence↗
PubMed1985

Binding of lectin-fluorescein conjugates to intracellular compartments of growth-plate chondrocytes in situ.

In this study, lectin-binding techniques are applied to growth-plate cartilage to analyze the intracellular localization of lectin-binding glycoconjugates of chondrocytes in situ. The binding of ten fluorescein-conjugated lectins is analyzed on 1-micron-thick Epon-embedded, nondecalcified sections of growth plates from Yucatan swine. Comparisons are made to intracellular binding in chondrocytes of tracheal, articular, and auricular cartilage. Ear epithelium, tracheal epithelium, and kidney are used as positive control tissues for the specificity of lectin binding. Only the mannose-binding lectins had affinity for the RER and nuclear envelope. Eight lectins reacted within the Golgi complex with characteristic patterns which ranged from localized fine linear strands to extensive vesicular accumulations. When cartilage slabs were exposed before embedment to the ionophore monensin to disrupt intracellular transport through the Golgi, it was possible to define differential subcompartments of the Golgi complex, based upon sites of sugar addition. Also, it was possible to characterize the cytoplasmic deposits of reserve-zone chondrocytes which were positive with concanavalin A as glycogen, based upon their sensitivity to amylase. This method allows resolution at the light-microscopic level of lectin-binding glycoconjugates with localization to specific organelles. Patterns of intracellular binding were consistent with biochemical data relating to the subcellular localization of processing steps of complex carbohydrates prior to secretion.

Animals↗
PubMed1987

Effects of hyperthermia, irradiation, and cytotoxic drugs on fluorescein isothiocyanate staining intensity for flow cytofluorometry.

Measurement of fluorescein isothiocyanate (FITC) staining intensity of cultured lymphoblastoid cells following hyperthermia showed large increases without concomitant increases in nuclear protein. Similar measurements of cells following incubation with cytotoxic drugs showed fluorescent intensity increases that exceeded the increases in nuclear protein that were due to the cell cycle blocking action of the drug. The reverse, however, was true for cells following irradiation. In contrast, FITC staining intensity and nuclear protein measurements of cells proceeding through the cell cycle after removal of the cycle blocking agent showed nearly parallel changes, although there were reproducible minor differences, especially following blocking with hydroxyurea. These results suggest that FITC staining intensity is a function not only of nuclear protein content but also of stain access to the reaction sites of the protein constituents of the chromatin. Thus, it is possible that FITC staining may be used as a probe of changes in chromatin structure following experimental manipulation of cells in vitro or treatment of tumors in vivo.

Antineoplastic Agents↗
PubMed1987

Spectroscopic properties of fluorescein in living lymphocytes.

Detailed studies have been performed on various spectroscopic properties such as time dependence and excitation wavelength dependence of the fluorescence anisotropy for fluorescein molecules introduced into rat thymus lymphocytes. Experimental results have been found to be well interpreted in terms of the coexistence of two types of dye molecules, i.e., free and bound molecules. The fluorescence spectrum of only the bound molecules has been obtained from the difference in the time-resolved spectra of fluorescence with two polarization directions. The time gate has been set at a sufficiently late time after the excitation, so that the polarization memories of the free molecules are lost. The spectrum thus determined agrees very well with that calculated from the spectral data in the stationary condition. From the above results, we come to the conclusion that the main factors which determine the fluorescence anisotropy inside the cell are the fraction and the anisotropy of the bound dye molecules. Finally, we discuss how these factors are related to biological quantities.

Animals↗
PubMed1989

Fluorescein isothiocyanate staining intensity as a probe of hyperthermia-induced changes in chromatin conformation.

In a previous report we presented evidence for large increases in fluorescein isothiocyanate (FITC) fluorescent intensity caused by hyperthermia which were not associated with synthesis of heat-shock proteins. We have now refined and considerably extended the measurements of increases in FITC fluorescent intensity caused by hyperthermia within the range 41.0 degrees C to 50.0 degrees C, and associated these with the extent of cell death caused by the hyperthermia. It appears that cell death ensues when the FITC fluorescent intensity has not returned to its baseline value within the time of one cell cycle. If thermotolerance is induced, there is a concomitant reduction in the increase in FITC staining intensity and the extent of cell death. When hyperthermia is followed by acid extraction, an additional increase in FITC staining intensity (above that due to hyperthermia alone) is observed, indicating separate sites of action on basic nuclear proteins. Hyperthermia and acid extraction have related effects on the relationship between FITC and propidium iodide staining. Hyperthermia-induced increases in FITC staining intensity are almost completely reversed by 6.7 mM formaldehyde with a marginal effect on the control FITC staining at this formaldehyde concentration. We suggest that hyperthermia causes extensive dissociation of basic protein-protein binding within nuclear chromatin, and that this may be a contributory cause of hyperthermia-induced cell death.

Cell Line↗
PubMed1991

Two- and three-color immunofluorescence using aminocoumarin, fluorescein, and phycoerythrin-labelled antibodies and single laser flow cytometry.

Antibodies coupled to 7-aminocoumarin (AMCA) emit a bright blue fluorescence under ultraviolet (UV) excitation and are therefore ideal for three-color immunofluorescence (IF) with fluorescein (FITC) and phycoerythrin (PE) labeled reagents; however, due to the different absorption spectra, the use of these fluorophores for multicolor flow-cytometric analysis requires a double light excitation source (e.g., two-laser system). We report a strategy which uses a single argon-ion laser to simultaneously excite AMCA, FITC, and PE, thus allowing the flow cytometric analysis of three immunological parameters. When the UV-visible argon-ion laser is fitted with an appropriate set of mirrors, the 35.1-363.8 nm (UV) and 488 nm wavelengths (accounting for 80 mW and 520 mW, respectively) are simultaneously generated; these lines can then be exactly focused on the same observation point by an achromatic cylindrical lens. A number of comparative analysis were performed with this instrumental set up to verify the sensitivity of AMCA IF and its possible application for multicolor immunophenotypic evaluation of blood cell subsets. When AMCA- and FITC-labeled antimouse Ig antibodies were assessed for their ability to detect limiting amounts of mouse monoclonal antibody bound to cells, the former was less sensitive than the latter. A number of factors, including differences in excitation energy (80 mW for AMCA and 520 mw for FITC) and extinction coefficients (1.9 x 10(4) for AMCA and 6 x 10(4) for FITC) could explain this result.(ABSTRACT TRUNCATED AT 250 WORDS)

Antibodies, Anti-Idiotypic↗
PubMed1983

The analysis of an anti-fluorescein cytotoxic response.

The detailed kinetics of the appearance of clones of anti-fluorescein cytotoxic T lymphocytes (CTL) in a primary limiting dilution response have been examined. The addition of factors from the supernatants of concanavalin A-stimulated rat spleen cell cultures was necessary to obtain linear dose-response curves and strong primary responses. In a standard primary response the maximum detectable number of specific clones was found, on day 5, to be derived from precursors having a frequency of 1 in 10 000 spleen cells. When limiting dilution cultures were restimulated on day 3, responses on day 5 were unaffected but new clones appeared on day 7 with a frequency of 1 in 1000. The emergence of the day-7 response depends on the addition of factor(s) on day 3. It is proposed that the early low frequency precursors detected on day 5 and the high frequency precursors detected on days 7-9 are derived from two stages in the differentiation lineage of CTL precursors. Restimulation of limiting dilution cultures involves not only the continued clonal expansion of primary responses but also the emergence of new "clones" of specific CTL.

Animals↗
PubMed1986

Binding of fluoresceinated epidermal growth factor to A431 cell sub-populations studied using a model-independent analysis of flow cytometric fluorescence data.

A method is developed for determining ligand-cell association parameters from a model-free analysis of data obtained with a flow cytometer. The method requires measurement of the average fluorescence per cell as a function of ligand and cell concentration. The analysis is applied to data obtained for the binding of fluoresceinated epidermal growth factor to a human epidermoid carcinoma cell line, A431. The results indicate that the growth factor binds to two classes of sites on A431 cells: 4 X 10(4) sites with a dissociation constant (KD) of less than or equal to 20 pM, and 1.5 X 10(6) sites with a KD of 3.7 nM. A derived plot of the average fluorescence per cell versus the average number of bound ligands per cell is used to construct binding isotherms for four sub-populations of A431 cells fractionated on the basis of low-angle light scatter. The four sub-populations bind the ligand with equal affinity but differ substantially in terms of the number of binding sites per cell. We also use this new analysis to critically evaluate the use of 'Fluorotrol' as a calibration standard in flow cytometry.

Carcinoma, Squamous Cell↗
PubMed1985

Analysis of 3-(2-(ethylamino)propyl)-1,2,3,4-tetrahydro-5H(1)benzopyrano (3,4-c)pyridin-5-one in plasma by liquid chromatographic column switching after derivatizing the secondary amine with fluorescein-6-isothiocyanate.

A high-performance liquid chromatographic (HPLC) assay has been developed to quantify the bronchodilator 3-(2-ethylamino)propyl)-1,2,3,4-tetrahydro-5H(1)benzopyrano(3,4-c)pyridi n-5-one (CI-923) (1) in human plasma. The drug and internal standard (2) were extracted from plasma (pH 10.5) with ether, and the secondary amines were derivatized with fluorescein-6-isothiocyanate (F6ITC) (3). The derivatives were isolated from the mixture by on-line HPLC column switching and were quantified with fluorometric detection (Ex: 484 nm; Em: 518 nm). Linearity was observed over the range 0.5-50.0 ng/mL of 1, and the recovery of drug from plasma was greater than 86%. The precision of the method was 3.7-5.2% RSD based on the analysis of seeded control pools containing 0.8, 20.0 and 40.0 ng/mL of 1. The accuracy of the method was +/- 3.3% relative error for the same three pools.

Bronchodilator Agents↗
PubMed1989

Diagnosis of gastric cancers with fluorescein-labeled monoclonal antibodies to carcinoembryonic antigen.

Forty gastric adenocarcinomas in 30 resected stomachs were examined by an endoscopic immunofluorescent technique using fluorescein isothiocyanate (FITC)-labeled antibodies to carcinoembryonic antigen (CEA). Fluorescence images of the tumors were obtained with a newly developed endoscopic television system for detecting faint fluorescence. Computer-assisted processing was used to enhance the detection and localization of the tumors visualized by the immunofluorescent technique. Twenty seven (90%) of 30 tumors showed positive fluorescence after treatment with FITC-labeled antibodies for 60 minutes, whereas only two (40%) of five cancers could be detected by the immunofluorescent technique after treatment with antibodies for less than 60 minutes. There was no significant relationship between positive fluorescence and the gross and histological types or the stages of the tumors. In contrast, no positive fluorescence could be demonstrated in cases of benign gastric lesions or after pretreatment with FITC-labeled alpha-fetoprotein. This study therefore suggests that the immunofluorescent technique using FITC-labeled anti-CEA antibodies can be useful in detecting the early stages of gastric cancer.

Adenocarcinoma↗
PubMed1994

Chemiluminescent and colorimetric detection of a fluorescein-labelled probe and a digoxigenin-labelled probe after a single hybridization step.

The objective of the simple and fast method we describe is the simultaneous hybridization of two non-radioactive probes and their detection from the same blot, using two different systems. These two probes are synthesized by PCR: one is labelled with fluorescein and the other with digoxigenin. The former is detected by chemiluminescence and the latter by colorimetry. We applied this rapid and simple method to the specific detection of multiplex polymerase chain reaction products. We used the human herpes simplex virus HSV1 and HSV2 PCR models studied in our laboratory.

Base Sequence↗
PubMed1991

Spectrophotometric analysis of sodium fluorescein aqueous solutions. Determination of molar absorption coefficient.

The usefulness of sodium fluorescein (SF) and related physical parameters were analysed. Two factors that may affect the molar absorption coefficient (epsilon) of this compound were the presence of impurities and the pH of the solution. As discrepant values can be found in the literature for that coefficient, a purification technique was used and SF quantification was performed according to sodium concentration determined by atomic absorption spectrophotometry. The molar absorption coefficient of the SF solution in phosphate buffer pH 7.4 was determined. To study the influence of pH on epsilon determination, absorption spectra at pH1, 3, 5, and 10 were also analysed.

Absorption↗
PubMed1995

Fluorescein as a label for non-radioactive in situ hybridization.

Non-radioactive techniques can be applied to many in situ hybridization (ISH) applications, and a number of non-radioactive labels for this process have been reported. However, these labels have some inherent problems in terms of both background and signal-to-noise values. We have sought to address these issues by searching for an alternative label that has the following features: efficient incorporation into probes, non-endogenous to biological systems, the availability of a high-affinity, high-specificity antibody. Fluorescein has been shown to meet these requirements. In addition, due to the fluorescent nature of the label, it has been possible to design a rapid, non-radioactive labelling assay and also to view in situ hybridization results by direct fluorescence in certain ISH applications. The hybridization kinetics have been investigated. Significant improvements have been made to the hybridization buffer leading to reduced background and increased rates of hybridization when compared to traditional hybridization buffers.

Animals↗
PubMed1994

Vascular permeability to sodium fluorescein in the rabbit cranial nerve root: possible correlation with normal cranial nerve enhancement on gadolinium-enhanced magnetic resonance imaging.

Vascular permeability in cranial nerve roots was examined after intravenous injection of sodium fluorescein in the adult rabbit. Fluorescence was observed in the distal nerves through the following portions: intracavernous portion of the oculomotor nerve, distal internal auditory canal segment of the facial nerve, and ganglionic portions of the trigeminal, glossopharyngeal and vagus nerves. In the acoustic nerve, the vestibular ganglion showed fluorescence. No fluorescence was observed in the olfactory or optic nerves. During in vivo gadolinium-enhanced magnetic resonance imaging (Gd-MRI) of two separate animals, trigeminal nerve enhancement was observed in the region showing fluorescence. Histologically, intense fluorescence was observed in ganglia and external nerve sheaths of the cranial nerves showing macroscopic fluorescence. A slight fluorescence was also seen in endoneurial connective tissue but not observed within the nerve fibers. The results of this study suggest that the physiological enhancement of human cranial nerves seen on Gd-MRI may correlate with vascular permeability.

Animals↗
PubMed1982

The three-dimensional reticular structure of the pig spleen demonstrated by labeling with fluorescein isothiocyanate.

Pig spleens were selectively perfused with a fluorescein isothiocyanate (FITC) containing medium. Histological sections of spleens excised one day later revealed the structure of the reticulum in thick cryostat sections directly in three dimensions. The pattern of the reticulum in silver impregnated sections was comparable to the FITC labelling pattern. This technique preferentially outlines the reticulum of the white pulp and the marginal zone of the spleen.

Animals↗
PubMed1992

Fluorescent staining of the actin cytoskeleton in human lymphocytes, monocytes and polymorphonuclear cells using a DNAse 1/anti-DNAse 1 immunoglobulin fluorescein conjugated system.

The actin associated with membrane-enriched extracts of leukocytes can be quantitated by DNAse 1 inhibition. Using this assay, we previously demonstrated that the actin level in monocytes was significantly higher than that in polymorphonuclear, T and B cells respectively. However, the extracellular location of the actin fraction detected by DNAse 1 inhibition (monomeric "G") remained unclear. This study using the DNAse 1/anti DNAse 1 immunoglobulin fluorescein conjugated system demonstrated that G-actin is present primarily in the cortical cell cytoplasm of leukocytes, in confirmation of our previous biochemical findings. Since the solubilized G-actin activities of membrane-rich lymphoid cell fractions, measured by DNAse 1 inhibition, are a reflection of the migratory potential, this immunofluorescent system may permit identification of the leukocytic cell subpopulations that have a potential for active circulation.

Actins↗