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PubMed1988

Increased intratumor concentration of fluorescein-isothiocyanate-labeled neocarzinostatin in rats under angiotensin-induced hypertension.

On the basis of the observation that the tumor tissue blood flow selectively increases under angiotensin (AT)-induced hypertension, the change of the drug concentration in the tumor and normal tissues was examined in male Donryu rats. The intratumor concentration of fluorescein isothiocyanate-labeled neocarzinostatin was about 2-fold higher in the AT-induced hypertension group than in the control up to 20 min after the drug injection. In the normal organs or the uninvolved organs of the tumor-bearing rats, however, no clear increase was seen in the experimental group compared with the control, as anticipated from the observation of the tissue blood flow. The present study supports the hypothesis that the enhanced anticancer effect in chemotherapy under AT-induced hypertension formerly reported is due to the tumor-selective enhancement of the drug delivery.

Angiotensin II↗
PubMed1991

Differential staining of human alpha beta and gamma delta T cells by the fluorescein conjugate of an anti-CD3 monoclonal antibody.

The enumeration of total T cells, an important function of the clinical immunology laboratory, utilizes antibodies to CD3, the macromolecular complex associated with the antigen-specific receptors of T cells. We compared the ability of some commonly employed commercial anti-CD3 reagents to stain human peripheral blood lymphocytes. Surprisingly, the fluorescein isothiocyanate (FITC) conjugate of Coulter clone T3 (FITC-T3) stained most T cells brightly, but selectively stained gamma delta T cells very dimly or not at all. In contrast, the other anti-CD3 reagents studied (FITC-Leu 4, PE-T3, PE-Leu 4, and indirectly labelled T3 and Leu 4) stained all T cells equivalently. Dual-colour flow cytometric analysis with FITC-T3 and PE-Leu 4 readily demonstrated a FITC-T3-/PE-Leu 4+ population of T cells. This unique population stained dimly or not at all with a combination of anti-CD4 and anti-CD8 monoclonal antibodies and positively with the pan-gamma delta T cell antibody TCR delta 1. Moreover, an excellent correlation was found between the number of FITC-T3-/PE-Leu 4+ cells and the number of TCR delta 1+ cells in 32 normal individuals. Thus, the FITC-T3-/PE-Leu 4+ phenotype accurately marks all gamma delta T cells. In contrast to FITC-T3, both PE-conjugated and unconjugated T3 stained gamma delta T cells brightly. Therefore, T3 binds to an epitope present on all T cells, but fluoresceinylation specifically attenuates this antibody's ability to bind to gamma delta T cells. These findings indicate that the use of FITC-T3 can result in a significant and variable underestimation of peripheral blood T cell number and demonstrate further that the CD3 complexes of human alpha beta and gamma delta T cells are significantly different.

Adult↗
PubMed1984

Dispersion of a fluorescein dye-ampicillin mixture after intratracheal administration in calves.

The pulmonary and systemic dispersion of a dye mixture after intratracheal (i.t.) administration was characterized in young dairy calves. Five calves were given i.t. injections of a fluorescein and ampicillin mixture, and were then killed at 2, 5 or 15 min after treatment. The respiratory system, nasal pharynx area, nose, liver, kidney, stomach and esophagus were removed and examined. Sections were taken from lung areas for histopathological examinations and all tissues were photographed, using floodlight and ultraviolet light. There was uniform low-grade fluorescence throughout the lung, but the greatest fluorescence followed an anterior ventral dispersion. The dye was readily absorbed from the lung into the bloodstream, causing fluorescence in the liver, bile, kidney and urine. Coughing of the calves during the injection caused some of the mixture to be expectorated and swallowed, resulting in fluorescence in the esophagus, stomach contents, and nasal pharanges, turbinates and nostrils.

Ampicillin↗
PubMed1983

Fluorescein-conjugated Bandeiraea simplicifolia lectin as a marker of endodermal, yolk sac, and trophoblastic differentiation in the mouse embryo.

The Bandeiraea simplicifolia lectin I (BSA-I) conjugated to fluorescein isothiocyanate was used as a histochemical reagent to study the mouse embryos from fertilization to early somitogenesis. No lectin binding could be detected on the embryonic cells in the preimplantation embryo. Lectin labeled intensely the zona pellucida. In the implanting embryos lectin binding was detected along the subtrophectodermal and Reichert's membrane, in the cytoplasm of the parietal and visceral endoderm, and the trophoblastic giant cells, but not in the ectodermal cells. Studies on explanted blastocyts cultured in vitro disclosed that the cytoplasmic BSA-I binding sites in trophoblastic cells develop gradually. In the 9-day somitic embryo BSA-I reacted with epithelial cells of the yolk sac, but not with the mesenchymal cells. A continuity between the lectin-reactive endoderm and the foregut epithelium could be demonstrated. These data indicated that BSA-I lectin can be used as a histochemical probe for endodermal (yolk sac) and trophoblastic differentiation in the peri-implantational mouse embryo.

Animals↗
PubMed1981

Effect of chloroquine and methylamine on endocytosis of fluorescein-labelled controlled IgG and of anti-(plasma membrane) IgG by cultured fibroblasts.

We report here the effect of chloroquine and methylamine two lysosomotropic drugs, on the binding, uptake and subcellular localization of fluorescein-labelled control immunoglobulin G (control IgG) a marker for non-specific adsorptive endocytosis and of anti-(plasma membrane) IgG (specific IgG), a specific ligand of cell-surface antigens. At 4 degrees C, methylamine and chloroquine inhibit the binding of control IgG to the cell surface, probably by a reversible competition. These two drugs, methylamine more than chloroquine, considerably slow down the rate at which control IgG is transferred from its binding sites on the phagosomal membrane to the lysosomal compartment; both drugs block almost completely the intralysosomal digestion of this IgG as well as the release of degradation products into the culture medium. They do not affect the binding and uptake of the specific IgG. In addition, methylamine seems to inhibit partially the return of the cell surface of membrane antigens and of membrane fragments bearing 5'-nucleotidase or binding sites for control IgG. We conclude that important steps (binding to cell surface, delivery to lysosomes, digestion and recycling of plasma membrane) involved in the uptake and the processing of IgG by fibroblasts are inhibited by these two substances. The effects of lysosomotropic agents on the regulation and function of the endocytic pathway and of lysosomes could have many pharmacological and therapeutic implications.

Animals↗
PubMed1988

Polymerization of actin modified with fluorescein isothiocyanate.

Solution properties of skeletal muscle actin, modified at lysine-61 with fluorescein isothiocyanate (FITC) [Burtnick, L.D. (1984) Biochim. Biophys. Acta 791, 57-62], were re-examined in this work by light scattering, analytical ultracentrifugation, fluorescence, electron microscopy and myosin ATPase activity measurements. Fluorescence measurements using trace amounts of actin labeled with N-(1-pyrenyl)iodoacetamide showed that the FITC modification inhibited but did not block completely the polymerization of actin by KCl and MgCl2. Sedimentation velocity runs of FITC-actin, incubated with 100 mM KCl and 2 mM MgCl2, revealed the presence in these solutions of polymeric, oligomeric and monomeric species. The critical concentration for FITC-actin polymerization under these conditions was 12 microM. As judged by electron microscopy, FITC-actin polymers were similar to but generally shorter than standard F-actin filaments. Light scattering measurements indicated that FITC modification inhibited also the polymerization of actin by myosin subfragment 1 (S1) but the resulting complexes were indistinguishable from standard, decorated actin filaments. MgATPase measurements showed that FITC-actin, polymerized by preincubation with S1, activated the MgATPase activity of S1 while the monomeric labeled protein did not. Thus, in analogy to native actin, the activating function of FITC-actin depended on the formation of actin filaments. Results presented in this study suggest that the region around lysine-61 of actin plays an important role in actin-actin contact and is less crucial to actomyosin interaction.

Actins↗
PubMedDate not supplied

Distribution of glycoconjugates in human testis. A histochemical study using fluorescein- and rhodamine-conjugated lectins.

Differentiation in the seminiferous epithelium involves the orderly transformation of germ cells into spermatozoa. We have employed ten fluorescein- and rhodamine-labeled lectins to visualize distinctive changes in the distribution of carbohydrate containing compounds during spermatogenesis and noticed the increase in RCA I, PNA, SBA and HPA binding sites during germ cell differentiation, suggesting the appearance of certain galactose and N-acetylgalactosamine containing glycoconjugates. Besides, in the cytoplasm of all germ cell types the positive reactions with Con A, LCA, WGA, LPA and UEA I indicate the presence of mannose, N-acetylglucosamine, sialic acid and fucose containing glycosubstances. Developing acrosomes demonstrated binding sites for most lectins, and particular HPA binding glycoconjugates were expressed in the equatorial segment region of late spermatids and testicular spermatozoa. In addition, the characteristic staining patterns of other testicular compartments are described. Our results suggest that human germ cells are rich in various carbohydrate containing compounds and there are specific alterations in cellular glycoconjugates during germ cell differentiation.

Acrosome↗
PubMed1983

In situ labelling of bone marrow lymphocytes with fluorescein isothiocyanate for lymphocyte migration studies in pigs.

In normal young pigs, the femoral artery and vein were cannulated and after occluding other vessels to one hind leg they were connected to an extracorporeal perfusion system. Fluorescein isothiocyanate (FITC) was added to the perfusate to selectively label cells in the bone marrow. Large numbers (approximately 0.9 X 10(9] of labelled lymphocytes left the bone marrow of one leg within 1 d and migrated via the blood to the bone marrow in other bones, lymph nodes, spleen, Peyer's patches and even into the thymus. On average 1.7% of the lymphocytes in the blood and about 1% within the spleen were labelled. Peyer's patches and the thymus showed very low indices. Thus the bone marrow is an integral part of the migratory route of lymphocytes. Selective labelling of bone marrow cells in their normal microenvironment with FITC is a suitable method for studies of cell migration from the bone marrow.

Animals↗
PubMed1983

Quality control of fluorescein isothiocyanate-labeled reagents.

Fluorescein-labeled reagents are not amenable to the same precision of definition as are chemical substances. Nonetheless extensive experimental evidence shows that certain preparative requirements and selected measurable chemical parameters of the product are of considerable predictive value in determining whether a reagent will be suitable in use. Adherence to these criteria and their continuous reappraisal combined with performance assessment and information on stability are all essential to ensure that reagents are of a quality acceptable to laboratory workers and legislative bodies. The need for manufacturers to document each procedural step and record the results is an essential element of good manufacturing practice now imposed by regulatory bodies. All reputable manufacturers adhere to these requirements, which serve not only to maintain standards but also to form a basis for corrective action.

Antibodies, Anti-Idiotypic↗
PubMed1977

Fluorescein and indocyanine green fluorescence angiography in study of affected males and in female carriers with choroidermia. A preliminary report.

26 males and 13 female carriers of different ages with choroideremia of varying severity were investigated using sodium fluorescein (FAG) and/or indocyanine green (ICG) fluorescence angiography. Females with minor changes present in pigment epithelium amy stay unchanged throughout life or gradually develop into a more advanced stage resembling the fundus picture of severely affected males. In moderately affected females there is a patchy degeneration of pigment epithelium in the macula. Peripapillary degeneration is seen also in indocyanine green fluorescence angiograms. In males, atrophic areas and the remaining choriocapillaris are clearly demonstrated in FAG and less well visible in ICG angiograms. ICG angiograms show choroidal vessels more clearly in cases where the pigment epithelium and the choriocapillaris are still present. In advanced cases in males and females, the choroidal blood circulation is slow.

Adolescent↗
PubMed1993

Assessment of tear drainage by fluorescein dye disappearance test after experimental canalicular obstruction.

Controversy exists about the role of upper and lower canaliculi in tear drainage. Fluorescein dye disappearance test was used to evaluate tear drainage after experimental obstruction of upper, lower, neither or both canaliculi in 18 subjects by hydroxypropil cellulose rods. When both canaliculi were occluded marked impairment was noted in all subjects (p < 0.05). Monocanalicular obstruction (either upper or lower) generally resulted in minimal impairment in most of the cases. Only 11% of upper canalicular obstruction and 20% of lower canalicular obstruction resulted in marked impairment. There was no significant difference between upper and lower canalicular obstruction (p < 0.05). In this study it is concluded that both upper and lower canaliculus have equal importance in tear drainage.

Adult↗
PubMed1982

Fluorescence microscopy: reduced photobleaching of rhodamine and fluorescein protein conjugates by n-propyl gallate.

n-Prop]yl gallate (0.1 to 0.25 molar, in glycerol) reduces by a factor of 10 the rate of fading of fluorescence of cell structures labeled with tetramethylrhodamine or fluorescein-conjugated antibodies. Hence, prolonged photographic exposure of immunofluorescently labeled cells in the fluorescence microscope yields images with increased sensitivity, making feasible multiple data collection, as with serial optical sectioning.

Fluorescein↗
PubMed1988

Rapid and simple method for double staining of bacteria with 4',6-diamidino-2-phenylindole and fluorescein isothiocyanate-labeled antibodies.

Bacteria were either heat fixed on microscope slides or filtered with 0.2 micron-pore-size Nuclepore filters. The samples were stained with 4',6-diamidino-2-phenylindole (DAPI) for total staining and with polyvalent rabbit antibodies and fluorescein isothiocyanate-conjugated swine anti-rabbit antibodies for specific staining. By switching between two different optical filter packages in the microscope, only one sample was needed for determining both total and specific counts of bacteria. False-positive counts and other artifacts that occur with antibody staining were easily distinguished when individual fluorescent particles were checked for DAPI fluorescence. The method for applying the general stain to membrane filters was performed quickly and simply by using a DAPI-soaked polypropylene filter that lay beneath the Nuclepore filter which collected the sample.

Animals↗
PubMed1985

Confirmation of Legionella pneumophila cultures with a fluorescein-labeled monoclonal antibody.

We compared a fluorescein-labeled monoclonal antibody directed against an outer membrane protein of Legionella pneumophila (Genetic Systems Corp. [GSC], Seattle, Wash.) with a similarly labeled polyclonal reagent (L. pneumophila serogroups 1 to 6, poly; BioDx, Inc., Denville, N.J.) for the confirmation of L. pneumophila isolates grown in culture. Duplicate suspensions of 52 organisms, including 21 L. pneumophila and 8 non-L. pneumophila species of legionella, were placed on individual glass slides, fixed, and stained with both reagents, and the results were compared. Both antisera correctly identified all L. pneumophila serogroups 1 to 6, but only the GSC reagent produced definitive staining of the L. pneumophila isolates of serogroups 7, 8, and 9. Additionally, the GSC reagent produced more uniform staining patterns around the legionella bacilli and displayed little background fluorescence when compared with the BioDx reagent.

Antibodies, Bacterial↗
PubMed1986

Comparison of cross-staining reactions by Pseudomonas spp. and fluorescein-labeled polyclonal and monoclonal antibodies directed against Legionella pneumophila.

Commercially prepared polyclonal antisera to Legionella pneumophila are known to cross-react with organisms of the genus Pseudomonas. To determine whether a commercially available monoclonal antibody reagent specific for L. pneumophila would also cross-react with pseudomonads, a two-laboratory study was undertaken to test both monoclonal and polyclonal reagents against 33 isolates of Pseudomonas spp., including 25 Pseudomonas aeruginosa, 4 P. putida, 2 P. maltophilia, 1 P. fluorescens, and 1 P. alcaligenes. Four antisera were tested; polyclonal anti-legionella antisera pools A and B (Centers for Disease Control [CDC], Atlanta, (Ga.), polyclonal 1-6 antisera (BioDx, Inc., Denville, N.J.), and a monoclonal antibody reagent produced by Genetic Systems Corp., Seattle, Wash. All reagents were labeled with fluorescein. Cross-staining reactions were found with the BioDx L. pneumophila antisera and 10 isolates of Pseudomonas. Four of these isolates demonstrated cross-staining with CDC pool A. When tested with individual serotype-specific reagents (CDC), three of four cross-reacted with L. pneumophila serotype 1 antisera; the fourth cross-reacted with serotype 3. No cross-staining reactions were noted with the monoclonal reagent and any of the pseudomonads tested, demonstrating that the Genetic Systems Corp. monoclonal reagent is the most specific of the four reagents tested.

Antibodies, Bacterial↗
PubMed2005

Lower limits of fluorescein and indocyanine green dye for digital cSLO fluorescence angiography.

BACKGROUND: With the advent of digital confocal scanning laser ophthalmoscopy it is possible to detect low levels of fluorescence. Here we used a novel confocal scanning laser ophthalmoscope (cSLO) to determine lower limits of dye required for fluorescein (FL) and indocyanine green (ICG) angiography. METHODS: A cSLO (Heidelberg retina angiograph 2, Heidelberg Engineering, Dossenheim, Germany) with an optically pumped solid state laser (488 nm) for FL and a diode laser (790 nm) for ICG angiography (FL/ICG-A) was used. 62 FL-As were performed in 53 patients and 45 ICG-As were performed in 39 patients with neovascular age related macular degeneration. The volume and overall dye content of bolus injections was gradually tapered (FL: 500 mg, 250 mg, 200 mg, 166 mg, 100 mg; ICG: 25 mg, 20 mg, 15 mg, 10 mg, 5 mg, 2.5 mg), while dye concentrations were kept constant at 100 mg/ml for FL and at 5 mg/ml for ICG. Images were obtained 1, 5, 15, and 30 minutes after dye injection. Image quality was evaluated by two independent readers using standardised criteria. RESULTS: For amounts down to 166 mg for FL and to 5 mg for ICG, sufficient image quality was achieved during all phases following injection. Only late phase images showed less contrast compared to typically used dye amounts, which was irrelevant for interpretation and clinical management. CONCLUSIONS: With the increased sensitivity of this novel cSLO system, amounts of injected dye during FL-A can be reduced to one third for FL and to one fifth for ICG without relevant loss of image quality or information compared to conventionally used dye levels. These amounts can be used for routine angiography and allow relevant savings for units performing FL-A.

Aged↗
PubMed2002

Diabetic macular oedema: the effect of photocoagulation on fluorescein transport across the blood-retinal barrier.

BACKGROUND/AIM: The visual loss secondary to diabetic macular oedema can be controlled to some extent by photocoagulation, though the mechanism of action is largely unknown. The purpose of the present study was to quantitate the effect of photocoagulation on the blood-retinal barrier using fluorescein as a tracer of passive and active transport. METHODS: A prospective study of 46 eyes in 34 patients with clinically significant macular oedema (CSMO) examined by vitreous fluorometry before and 6 months after macular photocoagulation treatment. RESULTS: In 23 eyes CSMO was not present at follow up (responding eyes), in another 23 other eyes CSMO was still present (non-responding eyes). With reference to the presence or absence of CSMO at follow up, the passive transport (permeability) for responding eyes decreased after photocoagulation in contrast with an increase in non-responding eyes; the difference between the groups at follow up was significant (p=0.03). The active transport for responding eyes decreased slightly at follow up, while it increased for non-responding eyes; the difference between the groups at follow up was not significant (p=0.09). CONCLUSION: Following photocoagulation a reduction of diabetic macular oedema, defined as disappearance of CSMO, is paralleled by a decrease of the passive permeability while the hypothesis of an increase in the active transport from the retina to the blood could not be supported by this study.

Adult↗