Fluorochrome Dyes Research Articles

Nuclear organization studied with the help of a hypotonic shift: its use permits hydrophilic molecules to enter into living cells.

A new procedure for introduction of hydrophilic molecules into living cells based on efficient uptake of these molecules into the cells during hypotonic treatment is presented and its use is demonstrated by a variety of applications. Experiments with cultured vertebrate and Drosophila cells and various animal tissues demonstrated that the increase in cell membrane permeability under hypotonic conditions is a general phenomenon in all animal cells tested. The efficiency of the method depends on the composition and temperature of the hypotonic buffer, the duration of the hypotonic treatment and the molecular weight of the molecules introduced into living cells. The versatility of this approach is demonstrated with various types of molecules such as modified nucleotides, nucleotides with conjugated fluorochrome, peptides, phosphatase substrates and fluorescent dyes. The method opens new possibilities for the direct investigation of a variety of biological problems as documented here with data on the functional organization of the cell nucleus.

Targeted cytoplasmic irradiation with alpha particles induces mutations in mammalian cells.

Ever since x-rays were shown to induce mutation in Drosophila more than 70 years ago, prevailing dogma considered the genotoxic effects of ionizing radiation, such as mutations and carcinogenesis, as being due mostly to direct damage to the nucleus. Although there was indication that alpha particle traversal through cellular cytoplasm was innocuous, the full impact remained unknown. The availability of the microbeam at the Radiological Research Accelerator Facility of Columbia University made it possible to target and irradiate the cytoplasm of individual cells in a highly localized spatial region. By using dual fluorochrome dyes (Hoechst and Nile Red) to locate nucleus and cellular cytoplasm, respectively, thereby avoiding inadvertent traversal of nuclei, we show here that cytoplasmic irradiation is mutagenic at the CD59 (S1) locus of human-hamster hybrid (AL) cells, while inflicting minimal cytotoxicity. The principal class of mutations induced are similar to those of spontaneous origin and are entirely different from those of nuclear irradiation. Furthermore, experiments with radical scavenger and inhibitor of intracellular glutathione indicated that the mutagenicity of cytoplasmic irradiation depends on generation of reactive oxygen species. These findings suggest that cytoplasm is an important target for genotoxic effects of ionizing radiation, particularly radon, the second leading cause of lung cancer in the United States. In addition, cytoplasmic traversal by alpha particles may be more dangerous than nuclear traversal, because the mutagenicity is accomplished by little or no killing of the target cells.

Simultaneous differentiation and quantitation of erythroblasts and white blood cells on a high throughput clinical haematology analyser.

After the neonatal period, the presence of nucleated red blood cells (NRBC) in peripheral blood is indicative of pathology. Despite the clinical utility of such measurements, automated NRBC counting has hitherto not been available on routine automated blood cell counting analysers. To address this, an automated method for the analysis of NRBC was developed and incorporated into the Abbott Cell Dyn 4000 (CD4000) haematology analyser. The system white blood cell (WBC) reagent was specifically formulated to preserve concomitantly white blood cell (WBC) morphology, rapidly lyse red blood cell and NRBC membranes, and subsequently stain NRBC nuclei with a nucleotide specific fluorochrome dye (Kim et al. 1996a). The fluorochrome itself does not permeabilize the membrane of intact viable white blood cells. The sample is processed by flow cytometry and the signals generated from an argon-ion laser light source are analysed. Axial light loss (AxLL), intermediate angle light scatter (IAS) and red fluorescence (FL3) are used to discriminate between particles of various types. By using these discriminators in a three-dimensional approach, NRBC from a discrete cluster which can easily be separated from leucocytes and enumerated as a distinct cell population during the optical WBC differential analysis. Consequently, accurate absolute WBC counts and differentials can be obtained even in the presence of NRBC. Background 'noise' (both fluorescent and non-fluorescent) from platelets. Howell-Jolly bodies, basophilic stippling, RNA from lysed reticulocytes, and DNA from leucocyte and megakaryocytic fragments are essentially eliminated (Kim et al. 1996b). While the membranes of intact and viable leucocytes remain impermeable to the passage of the fluorochrome stain, leucocytes with damaged membranes are permeable to the dye and generate FL3+ signals. Such cells, which are commonly seen as a consequence of sample ageing as well as in some distinctive pathologies, are identified by the algorithm (using their AxLL signal size) and are labelled as non-viable. Moreover, because non-viable leucocytes are retained in the WBC count and differential analyses, the CD4000 is further able to provide both numerical and graphical data regarding the relative frequency of viable and non-viable components. This additional information can serve as valuable 'decision-drivers' in the laboratory data review process.

Identification of calcium channels involved in neuronal injury in rat hippocampal slices subjected to oxygen and glucose deprivation

The presynaptic Ca 2+-influx affecting glutamate release during neuropathological processes is mediated via voltage-sensitive calcium channels (VSCCs). There is controversy, however, over the fractional contribution of the specific channel types involved. We have addressed this by investigating the protective effects of various VSCC blockers on oxygen and glucose-deprived rat hippocampal slices. The viability of treated and non-treated slices was assayed electrophysiologically by measuring the evoked population spike (PS) amplitude in the stratum pyramidale of the CA1 region and by imaging slices loaded with fluorochrome dyes specific for dead (ethidium homodimer) and live (calcein) cells using confocal microscopy. PS amplitudes were significantly ( P<0.01) depressed from 4.4±0.2 mV ( n=38) to 0.2±0.1 mV ( n=40) after the deprivation insult. Responses from deprived slices treated with ω-conotoxin MVIIC (100 nM; 4.2±0.5 mV; n=20) were not significantly different from control, non-deprived slice responses. In contrast, deprived slices treated with either L-type (0.1 or 1 μM nimodipine) or N-type (0.1 or 3 μM ω-conotoxin MVIIA) blockers showed no significant protection. The viability of CA1 neurons as revealed by the fluorescence live/dead confocal viability assay was consistent with the electrophysiological measurements. By comparison with previous studies using P- and Q-type blockers to attempt neuroprotection against the same deprivation insult, the rank order in which specific Ca 2+-channel types contribute to neuronal death due to oxygen and glucose deprivation was determined to be Q>N>>P>L. © 1997 Elsevier Science B.V. All rights reserved.

The Purity of Fluorescent Dyes: a Report Delivered to the Scientific Session of the Annual Meeting of the Biological Stain Commission

The Biological Stain Commission occasionally has been requested to certify fluorochromes as biological stains. Although formal certification is unlikely in the near future, the Commission is nevertheless concerned with the quality of these reagents. Commercial samples of fourteen fluorochromes were investigated for the presence of fluorescent organic impurities using reverse phase thin layer chromatography. Our findings suggest that some fluorochrome dyes are pure, but most are impure. Most fluorochromes vary in purity among vendors and among batches sold by single vendors. Impurities may be present at such high concentration that little of the presumed compound is present. Some impurities behave quite differently from the nominal dye. This may either create confusion or it might be useful. In the latter case, however, the impurity may occur only in a single batch. Impurities result from problems related to organic syntheses, separations, and economics. Solving those problems is often expensive, and what is expensive may not be performed. Fortunately, knowledge of synthetic chemistry often permits identification of fluorochromes likely to be impure. Moreover, predictions of likely staining effects of particular impurities can be made if appropriate structure-activity models are available. Possible actions by the Commission aimed at limiting the problems resulting from impurities of fluorescent dyes are noted.

Some events of mitosis and cytokinesis in the generative cell of Ornithogalum virens L.

The organization of the microtubule (Mt) cytoskeleton during mitosis and cytokinesis of the generative cell (GC) in Ornithogalum virens L. (bicellular pollen type, chromosome number, n = 3) from prophase to telophase/sperm formation was investigated by localization of α-tubulin immunofluorescence using a conventional fluorescence microscope and a confocal laser scanning microscope. Chromosomes were visualized with DNA-binding fluorochrome dyes (ethidium bromide and 4′6-diamino-2-phenyl-indole). The GC of O. virens is characterized by G2/M transition within the pollen grain and not in the pollen tube as occurs in the majority of species with bicellular pollen. It was found that prophase in the GC starts before anthesis and prometaphase takes place after 10 min of pollen germination. The prophase Mts are organized into three prominent bundles, located near the generative nucleus. The number of these Mt bundles is the same as the number of GC chromosomes, a relation which has not previously been considered in other species. The most evident feature in the prophase/ prometaphase transition of O. virens GC is a direct rapid rearrangement of Mt bundles into a network which appears to interact with kinetochores and form a typical prometaphase Mt organization. The metaphase chromosomes are arranged into a conventional equatorial plate, and not in tandem as is thought to be characteristic of GC metaphase. The metaphase spindle consists of kinetochore fibres and a few interzonal fibres which form dispersed poles. Anaphase is characterized by a significant elongation of the mitotic spindle concomitant with the extension of the distance between the opposite poles. At anaphase the diffuse poles converge. Cytokinesis is realized by cell plate formation in the equatorial plane of the GC. The phragmoplast Mts between two future sperm nuclei appear after Mts of the mitotic spindle have disappeared.

Application of confocal microscopy to exospore ultrastructure in megaspores ofSelaginella convoluta andS. marginata

Information pertaining to the arrangement of the rodshaped units that form the exospore ofSelaginella convoluta (Walk. Arn.) Spring andS. marginata (Humb. & Bonpl.) Spring megaspores was obtalned using both a confocal laser scanning microscope and a transmission electron microscope. Units are helically coiled, as we interpret them. The highest levels of fluorescence with confocal microscopy were in the places where the coiled exospore units that contain the fluorochrome dye overlap. These sites of overlap occurred in a close packed (hexagonal or pentagonal) arrangement. A more-or-less circular central nonfluorescent area was contiguous between overlapping exospore units. We conclude that the space between exospore units is a continuous channel (conduit). Each conduit is embraced by five or six helical-units that interdigitate.

ヒト口腔へん平上皮癌細胞株における抗癌剤によるアポトーシスの誘導

Although the primary target sites have been identified for many anticancer agents, little is known about the mechanisms by which anticancer agents cause the selective death of cancer cells. Squamous cell carcinoma, a major cancer in the head and neck region, is often resistant to anticancer agents. To investigate the mechanisms by which anticancer agents act cytotoxically on squamous cell carcinomas arising in the head and neck region, the effects of carboplatin (CBDCA), 5 -fluorouracil (5 -FU), or both on NA cells, a cancer cell line established from a patient with squamous cell carcinoma of the tongue, were examined. Although both anticancer agents, either alone or in combination, exerted cytotoxic effects against NA cells, no additive or synergistic effect was observed. The cytotoxic effects of these agents were mediated by apoptotic DNA damage of NA cells. Evidence for this was provided by the specific pattern of internucleosomal DNA fragmentation, as detected by electrophoresis, specific ELISA, and H33258 DNA specific fluorochrome dye. These results indicate that the cytotoxic effect of CBDCA or 5 -FU might be mediated by the process known as apoptosis. Furthermore, Northern blot analysis revealed that c-myc mRNA expression was down-regulated by both anticancer agents.

Remembrance of Professor Marcel Bessis (1917-1994)

After the introduction of analog video-capture technology, the light microscopy technique was replaced by computer-based digital image analysis, then by time-lapse video microscopy. These developments allowed to visualize longer periods of growth and movement of cells. The major hindrances of long-term scanning systems remained the limited period of time and nonphysiological conditions. Our multi-cell targeted time-lapse imaging system consists of integrated and motorized inverse microscopes located inside a CO2 incubator under optimized growth conditions, CCD cameras, illumination, steering electronics, computer and remote control. Each microscope is equipped with a high-sensitivity video camera connected to a dual image acquisition computer system operating with near-infrared light emitting diodes synchronized with image acquisition periods. Phototoxicity has been minimized by omitting fluorochrome dyes and using cold light-emitting diodes. Exposures are taken every minute and beside the analysis of individual slides, the whole series of photographs are converted to a video film by speeding up visualization to 30 exposures per second. During longer incubations, the medium is replaced by a pumping system introduced into the vials of the cell cultures. This protocol describes how microscope by time-lapse photography is used for the determination of cell cycle length. The protocol follows the cell growth and division of HaCaT cells through three cell cycles. The time of division helps to determine correctly the length of the cell cycle of individual cells. In contrast to other live-cell imaging approaches, our system has no time limit and cell growth can be traced for weeks uninterrupted.

Flow cytometric studies of equine phagocytes following strenuous exercise

SummaryThe objective of this study was to determine if strenuous exercise in unconditioned horses impaired function of neutrophils and alveolar macrophages. Flow cytometry was used to assess the effects of a single bout of strenuous exercise on these cells. Bronchoalveolar lavage and venous blood sampling were performed 30 min, 1 and 5 days after exercise. Exercise consisted of 2 min at 2,4 and 6 m/s and then 1 min at 1 m/s incremental speeds until exhaustion, on a level treadmill. Intracellular oxidation of the fluorochrome dye DCFH‐DA was stimulated by PMA and detected by a flow cytometer. Size and granularity of the 2 cell preparations were also measured. The respiratory burst induced in granulocytes was significantly inhibited 30 min after exercise (480 ± 182) compared to that prior to exercise (2359 ± 456). Inhibition at 1 and 5 days after exercise was less than at 30 min and did not differ significantly from either control values. No change in size or granularity was seen as a result of exercise. The alveolar macrophage population remained unchanged in size, granularity and fluorescent responses following exercise. Several cell samples taken before exercise exhibited peripheral blood eosinophilia. In these samples, granulocyte size was markedly reduced and DCFH‐DA oxidation was depressed; viability did not alter. This study suggests that strenuous exercise impairs the respiratory burst of granulocytes for several days. Other factors associated with peripheral eosinophilia also have suppressive effects on granulocytes.

Application of a flow cytometric method using autofluorescence and a tandem fluorescent dye to analyze human alveolar macrophage surface markers

Human resident alveolar macrophages (AM) exhibit autofluorescence when excited by light from 488 nm lasers used by most flow cytometers. Because this autofluorescence occurs at peak 540 nm, it obscures fluorescence generated by commonly used immunofluorescent reagent (e.g., antibodies conjugated to fluorescein isothiocyanate (FITC) or R-phycoerythrin (R-PE)) applied for cell surface marker analysis. Therefore, a two color flow cytometric method has been developed that permits the quantitative phenotypic analysis of AM without influence by their natural autofluorescence. In this method, a commercially available preparation of secondary polyclonal antibodies (recognizing primary specific mouse IgG monoclonal antibodies) that are conjugated to a tandem fluorochrome dye (containing R-PE and Cy5) is used. Using this method, the expression of 12 different surface markers on AM obtained from bronchoalveolar lavage (BAL) of 13 subjects was analyzed and compared with their expression on the surface of peripheral blood monocytes. This method will facilitate analysis of surface markers on AM in a variety of disorders.

Occurrence of G-banding in metaphase chromosomes of Encarsia berlesei (Hymenoptera: Aphelinidae)

Well-defined G-bands were obtained on somatic metaphase chromosomes of Encarsia berlesei using trypsin and warm 2x SCC in sequence. The G-banded pattern allowed rapid identification of all five metacentric chromosomes, which appeared uniformly lighted when stained with DAPI fluorochrome dye. It is stressed that ageing affects G-banding in this insect species; in fact, good banded chromosomes were obtained on 1-month air-stored chromosomes. Evidence for asynchronous condensation on the chromosomes of this species is also provided.

Physical ordering of three polymorphic DNA markers spanning the regions containing a tumor suppressor gene of renal cell carcinoma by three-color fluorescent in situ hybridization.

Fluorescent in situ hybridization (FISH) is a powerful technique for gene mapping and multi‐color FISH allows us to determine directly the order of two or more probes on both metaphase and interphase chromosomes. We report the physical ordering of three DNA markers by three‐color FISH using two fluorochrome dyes, fluorescein isothiocyanate (FITC; green) and rhodamine (red). The third color was visualized as a pseudo‐color (yellow) generated by optical interference with FITC and rhodamine. Using this system we could rapidly determine the order of three polymorphic DNA markers located on the 3p23–p21.2 bands which span a tumor suppressor gene for renal cell carcinoma, lung carcinoma, and several other types of tumors.

Variation of incorporation of fluorochrome dyes

Variation of incorporation of fluorochrome dyes

Quantitation of DNA and RNA in crude tissue extracts by flow injection analysis

An automated two-dye flow injection analysis system to quantitate DNA and RNA in curde extracts of tissues is described. The method uses the fluorochrome dyes ethidium bromide and Hoechst 33258. DNA concentration is determined directly from its fluorescence in Hoechst dye. RNA is estimated from fluorescence in ethidium bromide after subtraction of the fluorescence due to DNA. This method has several advantages: a simple extraction procedure, a low detection limit (0.01 μg DNA and 0.10 μg RNA), automation, and a high sample throughput.

Differential nuclear staining and cell count of trophectoderm and inner cell mass of bovine blastocyst fertilized in vitro by double fluorochrome dye technique.

二重蛍光染色法を用い,完全体外培養系により作出された牛胚盤胞期胚の栄養外胚葉(TE)と内細胞塊(ICM)を分別染色し,両細胞の割合を発生段階別,品質別に比較した。まず,家兎抗牛脾臓細胞血清を作製し,補体と共にTEに傷害を与え,ヘキスト33342とヨウ化プロピジウム(PI)による二重蛍光染色によりTEとICMの分別染色を行った。さらに完全体外培養系により作出された各発生段階の胚盤胞期胚を用いて,TEとICMの細胞数を個別に計測し,両者の比率を算出した。抗体と補体により細胞傷害を受けたTEはヘキストとPIによりピンクに,傷害を受けなかったICMはヘキストによりライトブルーに,それぞれ分別染色された。本法により計測した初期胚盤胞期胚の総細胞数は平均43±18で,総細胞数に対する割合は,TE:84.2±7.8%, ICM:15.8±7.8%であった。拡張胚盤胞期胚では,総細胞数:71±26で,総細胞数に対する割合は,TE:85.1±6.9%,ICM:14.9±6.9%であった。また脱出胚盤胞期胚では,総細胞数:108±37で,総細胞数に対する割合は,TE:88.1±5.6%, ICM:11.9±5.6%であった。以上の結果より,本法を用いて牛胚盤胞期胚のTEとICMの細胞数の分別計測を行うことは可能であることが明らかになった。また総細胞数は胚盤胞の拡張と透明帯からの脱出に伴い増加したが,ICMの割合は拡張期から脱出期にかけて逆に低下する傾向が認められた。

The influence of muscle flap coverage on the repair of devascularized tibial cortex: an experimental investigation in the dog.

Segments (2 cm) of canine tibial diaphyseal bone were devascularized and internally fixed with a plate. The medial cortex of the devascularized tibia was covered with skin in one experimental group (n = 7) and a local muscle flap in the other (n = 6). The animals were given intravenous fluorochrome dye and killed 42 days postoperatively. Enveloping callus formed around the cortex which was repaired by the formation of resorptive cavities on its external surface. New bone formation occurred within the resorptive cavities. Muscle flap coverage was associated with a sixfold increase in cortical porosity (p less than 0.005) and a fourfold increase in the area of enveloping callus (p less than 0.05). The area of intracortical new bone formation was greater in the cortex with muscle flap coverage (p less than 0.05). The maximum depth of intracortical new bone formation increased from 0.46 +/- 0.14 mm with skin coverage to 0.95 +/- 0.14 mm with muscle flap coverage (p less than 0.001). This study demonstrates that muscle flaps are superior to skin coverage in initiating the repair of devascularized cortical bone.

Fluorescence microscopy in the detection of early myocardial infarction

Fluorescent microscopy is amongst the many techniques devised for the post-mortem detection of early myocardial infarction. The method has the advantage of sensitivity, speed and simplicity, compared with more complex techniques such as enzyme histochemistry and electron microscopy. Disadvantages include the difficulty of permanent preservation of the sections and lack of sharp differentiation between normal and very early infarction — though the latter problem is common to most other methods, except enzyme histochemistry. There are two main techniques in fluorescent microscopy: (a) The examination of sections specifically stained by fluorochrome dyes; (b) The re-examination of haematoxylin-eosin sections for ‘autofluorescence’.

Human red blood cell membrane damage by endosulfan

Endosulfan is a chlorinated hydrocarbon insecticide. Its in vitro toxicity on human red blood cell membrane was studied by staining with a fluorochrome dye, merocyanine-540 (MC-540) and Scanning Electron muscopy (SEM). At a concentration of 0.001 μg/ml (1 ppb) endosulfan was found to damage human red cell membranes as demonstrated by fluorescence of 30–50% of red cells on staining with MC-540. This was supported by the finding of crenation and threading of red blood cells under SEM. At concentration of 1 μg/ml (1 ppm) the cells were markedly damaged.

Effect of Calcofluor white and Congo red on fungal cell wall morphogenesis: in vivo activation of chitin polymerization.

Calcofluor White (a fluorochrome dye) affected the growth of Geotrichum lactis by causing lysis of cells at the hyphal tips. This effect was prevented by the presence of an osmotic stabilizer. The growth of Saccharomyces cerevisiae was also affected, and multicellular aggregates were formed because of incomplete separation of mother and daughter cells; fluorescence microscopy indicated the presence of abnormally thick septa. The formation of rudimentary wall material by G. lactis protoplasts was promoted by Calcofluor or Congo red (another dye). The rate of chitin synthesis in protoplasts and growing cells was enhanced by both dyes. In contrast, both dyes inhibited chitin and beta (1,3)-glucan synthases in isolated cell-free systems. Degradation rates for beta (1,3)-glucan or chitin were not affected significantly by the dyes. Wheat germ agglutinin also affected chitin synthesis in protoplasts.