bluuebunny/crossref_metadata_2025_split huggingface dataset:
Computer image analysis of comparative genomic hybridization
Authors: Piper Jim , Rutovitz Denis , Sudar Damir , Kallioniemi Anne , Kallioniemi Olli‐P. , Waldman Frederic M. , Gray Joe W. , Pinkel Dan 
Published: June 2005

AbstractWe describe and evaluate the image‐processing and analysis techniques we have developed for the quantitative analysis of comparative genomic hybridization (CGH; Science 258:818, 1992). In a typical CGH application, two genomic DNA samples are simultaneously hybridized to metaphase chromosomes and detected with different fluorochromes. The primary data in CGH are contained in the intensity ratios of the fluorochromes as a function of position on the chromosomes, which reflect variation in DNA copy number ratio between the two DNA samples. Analysis involves chromosome segmentation, intensity normalization, background corrections, and calculation of the fluorescence intensity profiles and the ratio profile along the chromosome's length. Profiles from several copies of the same chromosome in different metaphases are averaged to reduce the noise. Confidence intervals are calculated and displayed for the mean profiles. The techniques were evaluated by examining the variability found in comparisons of two normal genomic DNAs, where the ratio was expected to be constant, and by measuring the ratios obtained for cell lines with cytogenetically documented copy number changes involving several chromosomal segments. The limits of sensitivity of CGH analysis were investigated by simulation. Guidelines for the interpretation of CGH data and indications of areas for future development of the analytical techniques are also presented. © 1995 Wiley‐Liss, Inc.
Quantitative analysis of comparative genomic hybridization
Authors: du Manoir Stanislas , Schröck Evelin , Bentz Martin , Speicher Michael R. , Joos Stefan , Ried Thomas , Lichter Peter , Cremer Thomas 
Published: June 2005

AbstractComparative genomic hybridization (CGH) is a new molecular cytogenetic method for the detection of chromosomal imbalances. Following cohybridization of DNA prepared from a sample to be studied and control DNA to normal metaphase spreads, probes are detected via different fluorochromes. The ratio of the test and control fluorescence intensities along a chromosome reflects the relative copy number of segments of a chromosome in the test genome. Quantitative evaluation of CGH experiments is required for the determination of low copy changes, e.g., monosomy or trisomy, and for the definition of the breakpoints involved in unbalanced rearrangements. In this study, a program for quantitation of CGH preparations is presented. This program is based on the extraction of the fluorescence ratio profile along each chromosome, followed by averaging of individual profiles from several meta phase spreads. Objective parameters critical for quantitative evaluations were tested, and the criteria for selection of suitable CGH preparations are described. The granularity of the chromosome painting and the regional inhomogeneity of fluorescence intensities in metaphase spreads proved to be crucial parameters. The coefficient of variation of the ratio value for chromosomes in balanced state (CVBS) provides a general quality criterion for CGH experiments. Different cutoff levels (thresholds) of average fluorescence ratio values were compared for their specificity and sensitivity with regard to the detection of chromosomal imbalances. © 1995 Wiley‐Liss, Inc.
Optical method for measurement of number and shape of attached cells in real time
Authors: Ramsden J. J. , Li S.‐Y. , Heinzle E. , Prenosil J. E. 
Published: June 2005

AbstractA novel, on‐line method of accurately parametrizing the shape of spreading cells is described. The substrate on which the cells are deposited serves as an optical waveguide. The method is based on the interaction between the evanescent part of the guided waves and the cells. No special labelling of the cells is required. A mathematical framework for interpreting the results is derived, and some illustrative results are presented. © 1995 Wiley‐Liss, Inc.
Study of surface carbohydrates on isolated Golgi subfractions by fluorescent‐lectin binding and flow cytometry
Authors: Guasch Rosa M. , Guerri Consuelo , O'Connor José‐Enrique 
Published: June 2005

AbstractThe Golgi complex is a functionally heterogeneous subcellular structure that plays a key role in the synthesis, maturation, and sorting of newly synthesized glycoproteins. Fluorescent lectins have been used extensively to analyze surface glycoproteins by flow cytometry in whole cells and more recently in isolated subcellular organelles, such as mitochondria and chloroplasts. We report here the use of several fluorescein‐isothiocyanate‐conjugated lectins to detect and quantify specific surface sugars by flow cytometry on isolated elements from purified cis and trans‐Golgi fractions from rat liver. Our results show that this approach may be useful to study Golgi composition and function, since it may reveal the intensity of specific binding of different lectins to each Golgi fraction and the percentage of elements binding the lectins specifically. Thus we show here that Golgi elements appear homogeneous in mannose and fucose, whereas galactose and N‐acetyl‐glucosamine residues are more abundant in the trans‐Golgi elements. © 1995 Wiley‐Liss, Inc.
DNA staining changes associated with apoptosis and necrosis in blood lymphocytes of individuals with HIV infection
Authors: Echaniz Pilar , de Juan Maria Dolores , Cuadrado Emilio 
Published: June 2005

AbstractWe used flow cytometry to quantitate cells that die by apoptosis or necrosis. The method uses low concentrations of two DNA binding dyes that allow one to establish selective regions for live, apoptotic, and necrotic cells in a rat thymocytes model. Quantitative analysis of blood lymphocyte death in individuals with HIV infection by this technique shows the presence of nonviable cells that exhibit a spectrum of changes in staining by DNA binding dyes. These changes range from typical features of cells undergoing programmed cell death or apoptosis to changes observed in cells that die by accidental death or necrosis. The proportion of cells exhibiting these lethal changes increases significantly in patients who progress to AIDS, but, although cells with staining features associated with apoptosis and necrosis were both found to be increased in in vitro‐activated cells from AIDS patients, spontaneous in vivo activation preferentially leads to apoptotic changes without a significant increase of cells exhibiting the staining changes associated with necrosis. © 1995 Wiley‐Liss, Inc.
Combined light and fluorescent microscopical imaging of nucleolar organizer regions and cellular rRNA as detected by fluorescent in situ hybridization
Authors: Pajor László , Honeyman Thomas W. 
Published: June 2005

AbstractA method for combined light and fluorescent microscopic imaging of nucleolar organizer regions and cellular rRNA is described. Nucleolar organizer regions were detected by silver staining (Ag‐NOR), and rRNA was detected by fluorescent in situ hybridization (FISH). MG‐63 human fibrosarcoma cells were silver stained prior to in situ hybridization. To quantitate Ag‐NOR within individual cells, brightfield images were digitized, and the total Ag‐NOR area/nucleus was determined. Fluorescent images were digitized, and the total cellular fluorescence was calculated after correction for nonuniformity of illumination. By using this method, it was shown that the Ag‐NOR procedure did not significantly affect the fluorescence intensity related to FISH. Furthermore, the hybridization procedure did not interfere with quantitation of Ag‐NOR. With this method, both Ag‐NOR and rRNA product can be quantitated within the same cell. Because the relationship of rRNA content to cell proliferation is well established, correlation to quantitative Ag‐NOR parameters within individual cells will contribute to the better definition of the relationship of quantitative Ag‐NOR indices with cellular proliferation. © 1995 Wiley‐Liss, Inc.
Cell membrane‐dependent chromatin condensation
Authors: Vinogradov Alexander E. 
Published: June 2005

AbstractIt is shown by means of flow cytometry that during several seconds after cell membrane damage by a non‐ionic detergent in physiologically relevant buffer solution, the chromatin of mouse thymocyte nuclei undergoes a drastic decondensation, which is revealed by a sharp increase of binding of DNA‐specific fluorochromes (olivomycin or propidium iodide) and of DNA accessibility to DNAse I digestion. A similar change is observed in dead cells. Roughly half of this decondensation can be prevented by lowering the pH of the outside medium to the level known to be inside the cells; the other half remains thus far unexplained (divalent cations and the difference between small anion species seem not to be involved). The approach is based on a novel observation that fixation by formaldehyde conserves chromatin structure before the action of detergent. Flow cytometric assay is proposed for monitoring these condensation/decondensation events in media of different composition. In addition, a new approach to viable/dead cell determination, which has the advantage of immediately fixing the cell state and preserving it for a reasonably long time, is proposed. © 1995 Wiley‐Liss, Inc.
Flow cytometric detection of micronuclei by combined staining of DNA and membranes
Authors: Wessels Jurina M. , Nüsse Michael 
Published: June 2005

AbstractA new staining method is presented for flow cytometric measurement of micronuclei (MN) in cell cultures and human lymphocytes using membrane‐specific fluorescent dyes in addition to DNA staining. Several combinations of fluorescent membrane and DNA dyes were studied for a better discrimination of MN from debris in a suspension of nuclei and micronuclei. For staining of membranes, the lipophilic dyes 2‐hydroxyethyl‐7,12,17‐tris(methoxyethyl)porphycene (HEPn) and 1,6‐diphenyl1,3,5‐hexatriene (DPH) were used in combination with ethidium bromide (EB), proflavine (PF), and Hoechst 33258 (HO). Due to their spectral properties, HO or EB combined with HEPn were not as suitable for the discrimination of MN from debris as was HEPn in combination with PF. With HEPn in combination with PF, however, additional noise was found at low fluorescence intensities, probably due to free fluorescent dye molecules in the solution. The optimal simultaneous staining of membranes and DNA was obtained using a combination of DPH and EB. The induction of MN in Chinese hamster and mouse NIH‐3T3 cells by UV‐B illumination was studied with this new staining technique. UV‐B illumination (280–360 nm) induced MN in both cell lines. Chinese hamster cells were found to be more sensitive to these wavelengths. Illumination with wavelengths above 360 nm did not induce MN in either cell line. The results obtained from human lymphocytes using the combination of EB and DPH were comparable to the results obtained with the combination of EB and HO. © 1995 Wiley‐Liss, Inc.
High‐speed photodamage cell selection using a frequency‐doubled argon ion laser
Authors: Keij Jan F. , Groenewegen Ad C. , Dubelaar George B. J. , Visser Jan W. M. 
Published: June 2005

AbstractA flow cytometer was developed for the high‐speed “sorting” of desired cells by selectively irradiating (zapping) the undesired cells from a population. After previous efforts to photoinactivate cells with photosensitizers had failed, it was decided to exploit the photosensitivity of the cell's DNA at 257 nm. It was shown that a 257 nm laser output power of 20–100 mW was sufficient to induce a 4.5 log cell kill after the cells were processed through a focused 257 nm laser beam. Experiments proved that the photodamage flow cytometer (ZAPPER) could selectively photoinactivate cells at rates over 22,000 events/s, and selection purities ranged from 81% to 100%.The yields of the desired cells depended on the selection mode. In the Enrichment mode, the zap laser was not aimed at the jet, and only undesired cells were exposed to a brief ultraviolet (UV) pulse after modulation of the UV laser beam. The yields of desired cells ranged from 95% to 105%. In the Purge mode, the zap laser beam was aimed onto the jet, and only desired cells were allowed to pass after deflection of the UV laser beam; the yields of desired cells ranged from 12% to 52%. The cause of the reduced yields in the PURGE mode was traced to the fact that the Electro‐Optic Modulator was used to modulate the zap laser proved too slow for the intended application.The lifetime of the frequency‐doubling crystal used for the generation of the 257 nm beam was found to be limited to several days. These technical limitations could theoretically be overcome by the application of large argon ion lasers capable of emissions at 257–275 nm and an Acousto‐Optic Modulator to deflect the zap laser. © 1995 Wiley‐Liss, Inc.
Modulation of membrane fluidity and lipidic metabolism in transformed rat fibroblasts induced by the sesquiterpenic hormone farnesylacetone
Authors: Rodes Jean‐François , Berreur‐Bonnenfant Josette , Trémolières Antoine , Brown Spencer C. 
Published: June 2005

AbstractFarnesylacetone is a natural terpene extracted from androgenic glands of the crustacean Carcinus maenas and is capable of inhibiting proliferation, notably in transformed mammalian cells. Flow cytometry with three lipophilic probes, diphenylhexatriene, trimethylammonium‐diphenylhexatriene, and Nile red, has revealed modifications of the lipidic metabolism in transformed FR3T3‐mTT4 rat fibroblasts treated by farnesylacetone, including changes in membrane fluidity. Farnesylacetone strongly increased the number of neutral lipidic droplets in the cytoplasm. Moreover, after prolonged terpene treatment, the membrane fraction of cells contained a substantial level of triglycerides. Farnesylacetone provoked an immediate but transitory increase in membrane fluidity of the cell membrane. The change in measured lipid fluidity appears to be due to these triglycerides rather than to the phospholipids. © 1995 Wiley‐Liss, Inc.
Comparison of cell viability probes compatible with fixation and permeabilization for combined surface and intracellular staining in flow cytometry
Authors: O'Brien Melissa C. , Bolton Wade E. 
Published: June 2005

AbstractDead cells represent a significant source of interference in the flow cytometric analysis of viable cells primarily due to nonspecific uptake of probes, increased autofluorescence, and altered antigen expression and DNA content. Traditional methods of dead cell exclusion, based on light scatter or uptake of dyes such as propidium iodide (PI) or fluorescein diacetate (FDA), are appropriate for the analysis of fresh, relatively homogeneous samples. However, they are incompatible with the development in this laboratory of a solid tumor monoclonal antibody panel incorporating combined surface and intracellular staining: Light scatter is unreliable in heterogeneous samples such as solid tumors, and most of the widely used viability probes are incompatible, due to weak or reversible binding, with the use of permeabilizing agents for intracellular staining. © 1995 Wiley‐Liss, Inc.To determine the best viability marker for inclusion in the solid tumor panel, we compared cultured cells held under hypoxic conditions for up to 15 days after harvest, stained with eight viability probes, and processed according to the solid tumor panel procedure (unprocessed cells from each day, stained with PI, were used as standards). The viability probes included PI (in processed and unprocessed samples); 7‐aminoactinomycin D (7‐AAD); TO‐PRO‐3; laser dye styryl (LDS)‐751; ethidium monoazide (EMA); and actin, cytokeratin, and tubulin indirectly labelled with sheep‐α‐mouse‐FITC (SAM‐FITC). The selection criteria for the best viability probe included broad cell type specificity: low nonspecific staining of live cells, specific staining of dead cells strong enough to withstand the permeabilization procedure, high signal‐to‐noise ratio throughout the time course, and compatibility with the four other fluorescent probes making up the tumor antibody panel. TO‐PRO‐3, LDS‐751, and PI (in processed cells) stained both live and dead cells indiscriminately. Actin‐SAM‐FITC, EMA, and 7‐AAD did not display sufficiently high signal‐to‐noise ratios over the entire time course. Cytokeratin‐SAM‐FITC was acceptable in every respect other than its specificity only for cells of epithelial origin. Tubulin‐SAM‐FITC alone satisfied all the criteria and was selected for inclusion in the monoclonal antibody panel as a viability probe. © 1995 Wiley‐Liss, Inc.
Limited loss of nine tumor‐associated surface antigenic determinants after tryptic cell dissociation
Authors: Corver Willem E. , Cornelisse Cees J. , Hermans Jo , Fleuren Gert Jan 
Published: June 2005

AbstractDisodium ethylenediaminetetraacetic acid (EDTA) or trypsin/EDTA are frequently used for the dispersion of monolayer cells into single cell suspensions allowing flow cytometric analysis of surface antigenic determinants. A disadvantage of EDTA is the slow action of this agent, whereas trypsin might affect the antigenic determinants under focus. We studied the possible deleterious effect of trypsin on three different ovarian carcinoma cell lines, COV413b, COV362.c14, and NIH:OVCAR‐3, on cell surface antigenic determinants by flow cytometry. Either EDTA or trypsin/EDTA was used for detachment and dissociation of monolayer ovarian cancer cell lines, followed by indirect immunofluorescence with a panel of monoclonal antibodies directed against nine different surface antigenic determinants, including six markers directed against widely distributed antigens. Compared to EDTA, trypsin/EDTA resulted in higher total cell yields and rapid detachment and dissociation into single cell suspensions with significantly lower amounts of dead cells detected by both trypan blue and propidium iodide (PI). Large differences in antigen expression were observed for the different cell lines. However, all antigenic determinants tested could still be detected after tryptic proteolysis. Three antigenic determinants were significantly decreased after trypsin/EDTA compared to EDTA detachment. CA 125 was decreased on COV362.c14 and NIH:OVCAR‐3 cells, respectively. BMA 180 and ICAM‐1 were decreased on COV413b cells. This cell line‐dependent decrease might be caused by differences in glycosylation. We conclude that trypsin/EDTA can be used for rapid monolayer cell detachment with high cell yields and limited loss of antigenic determinants tested. © 1995 Wiley‐Liss, Inc.
Computer software for testing drug susceptibility of malaria parasites
Authors: Reinders Paul P. , van Vianen Philip H. , van der Keur Maarten , van Engen Anneloes , Janse Chris J. , Tanke Hans J. 
Published: June 2005

AbstractA computer program is described for the automated analysis of data obtained by flow cytometry for in vitro antimalarial drug susceptibility testing. Samples of malaria‐infected red blood cells (RBC), which were cultured in the presence of different concentrations of antimalarial drugs, were stained with Hoechst. The Hoechst fluorescence intensity of infected RBC corresponds to DNA content of the parasites and to their stage of development. After measurement of the samples by a FACStar flow cytometer equipped with a UV laser and an autosampler, FCS 1.0 data files were generated. The HP PASCAL program developed for these files identifies five different populations—uninfected RBC, infected RBC, free parasites, leukocytes, and debris—on the basis of their light scatter and fluorescence characteristics. The program calculates the percentage of infected cells, the total number of parasite nuclei, and the average number of nuclei per parasite. The results of each culture are presented as a drug dose‐response curve. During data analysis, user interaction is limited to selecting the first file of the first culture. The algorithm then processes each culture automatically. Potential problems or difficulties in analysis are flagged. To date, a total of 862 drug tests have been evaluated and fall into two classes, an extended microtest and the World Health Organization standardized microtest. These tests gave satisfactory results in more than 99% of the cases. © 1995 Wiley‐Liss, Inc.
Mouse testis cell sorting according to DNA and mitochondrial changes during spermatogenesis
Authors: Petit J. M. , Ratinaud M. H. , Cordelli E. , Spanò M. , Julien R. 
Published: June 2005

AbstractFlow cytometry can measure variations in DNA content and chromatin structure as well as dramatic changes in the mitochondria of germ cells during maturation from spermatogonia to elongated spermatids. Using 10‐N nonyl acridine orange (NAO), an inner mitochondrial membrane dye, it is easy to follow mitochondria rearrangements. Mouse testis cells stained with the DNA fluorescent probe propidium iodide (PI) and analyzed by flow cytometry can be discriminated on the basis of their ploidy levels into five main regions corresponding to elongated spermatids, round spermatids, diploid, S‐phase, and tetraploid cells. The simultaneous use of PI and NAO demonstrated the presence of cells having low and high mitochondrial content in the haploid, diploid, and tetraploid compartments. Eleven sorting windows were selected from the bivariate analysis (PI/NAO) and the corresponding cells were identified by microscopic observation. Cells were also discriminated by two parameter analysis of DNA content vs. cell diameter. The definition of seven different regions allowed us to determine NAO or rhodamine 123 (Rh 123) uptakes in each compartment. We observed that the ratio (Rh 123/NAO) dramatically changed according to the progression of cell differentiation which occurs during spermatogenesis. © 1995 Wiley‐Liss, Inc.
Topical application of 12‐O‐tetradecanoylphorbol‐13‐acetate induces dyssynchronous expression of keratins K1 and K10 in mouse epidermis
Authors: Heyden Anders , Lützow‐Holm Claus , Clausen Ole Petter F. , Brandtzaeg Per , Huitfeldt Henrik S. 
Published: June 2005

Abstract12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) is a potent tumor promoter that causes severe alterations in the biosynthesis of epidermal keratins. This study shows that TPA induces a dyssynchrnous effect on keratin expression in stratified squamous epithelium. The effect of TPA on the separate expression of the maturation‐associated keratins K1 and K10 was studied by immunohistochemistry in an unperturbed replicative keratinocyte population of hairless mice epidermis in relation to changes in the cell cycle time during regeneration. Keratinocytes in DNA synthesis were pulse‐labeled by intraperitoneal injection of the thymidine analogue 5‐bromodeoxyuridine (BrdUrd) 1 h before a single topical application of TPA. The BrdUrd‐labeled cell cohort, representing an originally unperturbed replicative keratinocyte population exposed to TPA mainly in the postreplicative period, was followed for up to 97 h. The results suggested unaltered timing of the onset of K1 and K10 expression comparared with normal epidermis (18 and 24 h, respectively, following DNA synthesis). This indicates that the synthesis of both keratins was programmed before the keratinocytes entered their last DNA synthesis. A reduction in K10 expression from about 30 h compared with that of K1 expression was observed. Mathematical modeling suggested a delay in K10 expression related to the second and third rounds of cell divisions after pulse‐labeling. How TPA induces such dyssynchrony in K1 and K10 regulation remains unknown. © 1995 Wiley‐Liss, Inc.