bluuebunny/crossref_metadata_2025_split huggingface dataset:
Neutrophil function screenlng in patients with chronic granulomatous disease by a flow cytometric method
Authors: Epling C. L. , Stites D. P. , McHugh T. M. , Chong H. O. , Blackwood L. L. , Wara D. W. 
Published: August 2005

AbstractNeutrophils from patients with chronic granulomatous disease (CGD) fail to produce a significant oxidative burst following stimulation. We have evaluated the use of flow cytometry and the dye 2′,7′dichlorofluorescein diacetate (DCF) for routine screening for deficiencies of neutrophil oxidative burst. A range for DCF fluorescence for phorbol myristate acetate stimulated and non‐stimulated neutrophils was established based on data from 52 healthy adults. Samples from three patients with suspected neutrophil dysfunction, three patients with X‐linked CGD, and one patient with autosomal recessive (AR) CGD were evaluated with both the DCF assay and the quantitative nitroblue tetrazolium dye reduction (NBT) test. For the DCF test, the ratio of mean fluorescence intensity of stimulated to non‐stimulated neutrophils was <5 for CGD patients and from 16 to >50 for healthy individuals. With the DCF test, two populations of neutrophils could be identified in samples from four carriers of X‐linked CGD, although two carriers of AR CGD had NBT and DCF results in the normal range. Our data suggest the DCF test is a sensitive and convenient method for detecting CGD. © 1992 Wiley‐Liss, Inc.
Drop‐delay measurement using enzyme‐coated particles
Authors: Lazebnik Y. A. , Poletaev A. I. , Zenin V. V. 
Published: August 2005

AbstractA simple technique is suggested for the measurement of drop delay for flow sorting. While the flow cytometer was set to sort a fixed number of particles, the drop‐ delay setting was changed step by step, and at each step the HRP‐coupled particles were sorted into a well of an immunoassay strip. Then the HRP activity of the sorted samples was revealed by routine methods. The maximum level of the enzyme activity shows the proper drop‐delay setting. Determination of the drop‐delay setting takes only a few minutes. The technique is independent of the type of flow cytometer and does not require any additional equipment. © 1992 Wiley‐Liss, Inc.
Protooncogene expression in subpopulations of cells front leukemia patients
Authors: Hulette Ben C. , Banavali Shripad D. , Finke Daniel P. , Gopal Venu , Preisler Harvey D. 
Published: August 2005

Abstract
This report describes a method for pre‐serving the light scatter patterns of cells in which myc and myb expression are being measured. Exposure of cells to 1% paraformaldehyde for 72 h prior to antibody staining for myc and myb proteins preserved the light scatter patterns. Using this method, myc and myb expression was found to be highest in lymphocytes and monocytes and lowest in granulo cytes. The measurement of differences in the level of expression of these genes in subpopulations of leukemia cells obtained from individual patients is possible as is assessment of the levels of expression amongst normal and leukemia cells present in the same patient. © 1992 Wiley‐Liss, Inc.
Photo‐bleaching and photon saturation in flow cytometry
Authors: van den Engh Ger , Farmer Colleen 
Published: August 2005

AbstractIn flow cytometry, small particles travel at a high speed through a bright light spot. The high light intensity at the point of measurement causes measurable photon saturation. This observation indicates that the rate at which individual dye molecules emit photons is close to the maximum emission rate. Despite the short exposure time, individual molecules may go through a few hundred excitation cycles while they are in the light beam. The absorbed light dose causes significant dye destruction. This article presents experimental procedures to determine the extent of photon saturation and photo‐bleaching of dyes bound to cell nuclei in a flow cytometer. Measurements of Hoechst and propidium iodide bound to chromatin show that the amount of dye bleached per emitted photon is the same at low and high illumination intensities. This finding indicates that photon emission and dye destruction are both the result of the absorption of single excitation photons. The experimental observations allow rough estimates of the lifetime of the excited state and the lifetime of the molecule. The lifetime of the Hoechst 33258 bound to DNA is estimated to be 100 excitation‐relaxation cycles. The average propidium iodide molecule lasts ∼ 200 excitation‐relaxation cycles. The theoretical considerations show that the optimal illumination conditions are different for bleaching and nonbleaching dyes. Aoa optical arrangement for high precision measurements of bleaching dyes is presented. Published 1992 by Wiley‐Liss, Inc.
Signal transduction in monocytes and granulocytes measured by multiparameter flow cytometry
Authors: Lund‐Johansen Fridtjof , Olweus Johanna 
Published: August 2005

AbstractThe novel calcium indicator fura red and the oxidative burst indicator dihydrorhodamine (both excited at 488 nm) were used in combination with multiparameter flow cytometry to allow simultaneous kinetic measurements of calcium fluxes and oxidative bursts in monocytes and granulocytes. Using this method it was possible to obtain direct evidence for the following cell type‐ and stimulus‐specific differences in signal transduction pathways: (1) n‐formyl‐methionyl‐leucylphenylalanine (FMLP)/cytochalasin B‐induced oxidative burst is several‐fold higher in granulocytes than in monocytes although the calcium fluxes have similar amplitudes in the two cell types; (2) stimulus‐induced calcium fluxes in granulocytes are mainly due to release from intracellular stores, whereas monocytes mobilize calcium mainly by influx from the medium; (3) the FMLP/cytochalasin Binduced calcium flux in monocytes is less sensitive to the G‐protein inhibitor pertussis toxin than the flux in granulocytes; (4) in contrast to FMLP/cytochalasin B, the protein kinase C activator phorbol myristate acetate (PMA) induces an oxidative burst that is not preceded by a cytoplasmic calcium flux; (5) the PMAinduced oxidative burst can be triggered in monocytes and granulocytes that are depleted of intracellular calcium ions, whereas that induced by FMLP/cytochalasin B can not; (6) the G‐protein inhibitor pertussis toxin blocks an early event in the signal transduction pathway of FMLP/cytochalasin B, as shown by inhibition of both calcium fluxes and oxidative burst; and (7) 100 nM of the protein kinase inhibitor staurosporine blocks the FMLP/cytochalasin B‐induced respiratory burst by interfering with a step downstream to cytoplasmic calcium fluxes, whereas only 10–20 nM is necessary to block PMA‐induced oxidative burst. © 1992 Wiley‐Liss, Inc.
Swine chromosomal DNA quantification by bivariate flow karyotyping and karyotype interpretation
Authors: Schmitz Annette , Chaput Brigitte , Fouchet Pierre , Guilly Marie Noelle , Frelat Gerard , Vaiman Marcel 
Published: August 2005

AbstractHuman and swine chromosomes were analyzed separately and as a mix to obtain bivariate flow karyotypes. They were normalized to each other in order to use the human chromosomal DNA content as standard. Our results led to the characterization of the “DNA line” in swine identical to the human “DNA line.” Estimation of the DNA content in megabase pairs of the swine chromosomes is proposed. Chromosomal assignment to the various resolved peaks on the bivariate swine flow karyotype is suggested from the relation between DNA content quantified by flow cytometry and chromosomal size. Swine chromosomes 1, 13, 6, 5, 10, 16, 11, 18, and Y were assigned to peaks A, B, C, K, L, N, O, Q, and Y, respectively. Peaks D anal E were assumed to contain chromosomes 2 and 14, but without specific assignment. Similarly, P and M peaks were expected to correspond to chromosomes 12 and 17. Of the remaining chromosomes (3, 7, X, 8, 15, 9, and 4), chromosomes 3, 7, and X, which were assigned previously to peaks F, G, and H, respectively, led us to deduce that chromosomes 15 and 8 belonged to peaks I and J, and chromosomes 9, 4, and X to peak H. © 1992 Wiley‐Liss, Inc.
Comparative evaluation of fresh, fixed, and cryopreserved solid tumor cells for reliable flow cytometry of DNA and tumor associated antigen
Authors: van Dam P. A. , Watson J. V. , Lowe D. G. , Chard T. , Shepherd J. H. 
Published: August 2005

AbstractFive different protocols for the shortterm preservation of cells used for multiparameter flow cytometric assay of tumour associated antigens (TAA) and DNA were assessed in cell suspensions prepared by mechanical disaggregation of 15 gynecological tumors. The protocols at 4°C were (1) storage in buffer, (2) storage in 50% methanol, and (3) storage in buffer after formalin fixation. Tissues were also cryopreserved as cell suspensions and tissue blocks. When the TAA expression and DNA histograms of the preserved cells were compared with those in fresh cell suspensions, cryopreservation was found to be the best method: TAA expression was well preserved and there was a good correlation between TAA expression and the quality of the DNA histograms, respectively, in fresh and cryopreserved cells (RS: 0.82–0.91, P<0.001 for all TAAs). The cell suspensions preserved at 4°C all showed a significant increase in background fluorescence (P<0.05) and a reduction in the TAA specific fluorescence (P<0.011). Methanol fixation was better than buffered formalin for the proteins studied, though both gave significantly worse results than cryopreservation. The quality of these cell suspensions and the correlation with TAA measurements in fresh cell suspensions deteriorated progressively with time, particularly if they were stored more than a week. © 1992 Wiley‐Liss, Inc.
Flow‐cytometric enumeration of micronucleated polychromatic erythrocytes in mouse peripheral blood
Authors: Grawé Jan , Zetterberg Gösta , Amnéus Herman 
Published: August 2005

AbstractA flow‐cytometric assay is described that can be used to determine the frequency and the DNA content of mirronucleated polychromatic (PCE) and normochromatic (NCE) erythrocytes in mouse peripheral blood. Thiazole orange was used for discrimination between PCEs and NCEs, while Hoechst 33342 was used to detect micronucleated PCEs and NCEs. Up to 70,000 polychromatic erythrocytes can be analyzed in less than 10 min. This corresponds to 150–3,000 micronucleated polychromatic erythrocytes, 90–95% of which are true events as determined with a fluorescence microscope after sorting. Using X‐rays as the inducing agent in dose‐response experiments, a significant increase can be registered at doses of 0. 02 Gy. It seems possible that the method will also allow the detection of clastogenic effects of other inducing agents at lower doses than previously possible. © 1992 Wiley‐Liss, Inc.
Three‐dimensional image processing for morphometric analysis of epithelium sections
Authors: Albert René , Schindewolf Thomas , Baumann Irith , Harms Harry 
Published: August 2005

AbstractThe reproducible classification of poorly differentiated abnormal epithelium specimens is still a diagnostic problem. The computer‐aided method described here improves the differentiation between benign and malignant epithelium specimens. Hematoxylin and eosinstained sections of normal squamous epithelium, dysplasia, carcinoma in situ, and carcinoma were scanned in a TV microscope system and analyzed by means of image processing methods on a DEC 5000/200 workstation. From the 15–20 wm thick histological sections, 3–5 focus positions in steps of 1–4 μm were scanned. The segmentation of the cell nuclei was performed automatically by color analysis and geometric operations. For each nucleus the best focus level was selected and at this level the center of the cell was calculated. Graph theoretical methods were applied to analyze the morphometry of the epithelium specimens. The minimal spanning tree was computed in the three‐dimensional (3D) space of the sections with the selected centers of the nuclei as vertices. The best feature found for discrimination of the specimens is the average length of all edges in a tree. In the two‐dimensional (2D) analysis we had to accept an error probability of about 20% in differentiation of dysplasia and carcinoma. In contrast to this we differentiated normal squamous epithelium, dysplasia, and carcinoma with a correct classification rate of 100% in the 3D analysis. © 1992 Wiley‐Liss, Inc.
Simultaneous analysis of immunophenotype and apoptosis of murine thymocytes by single laser flow cytometry
Authors: Bruce Lyons A. , Samuel Kay , Sanderson Andrew , Maddy Alun H. 
Published: August 2005

AbstractThe study of the role of apoptosis in thymocyte development has been hampered by the lack of a means of directly immunophenotyping cells undergoing the early phase of apoptosis. This restriction has been overcome by single laser flow cytometry in which apoptosis is detected by Ethidium Bromide (EBr) staining and cell phenotype by binding of FITC‐labelled antibody. The initial phase of apoptosis is observed as a cell population that stains faintly with EBr preceding the characteristically bright EBrstaining normally associated with cell death. Here we directly demonstrate using single laser flow cytometry that CD4+ CD8+ CD3low/CD3ntermediate thymocytes undergo apoptosis in vitro in response to glucocorticoid treatment. © 1992 Wiley‐Liss, Inc.
Quantification of inter‐ and intra‐nuclear variation of fluorescence in situ hybridization signals
Authors: Nederlof P. M. , van der Flier S. , Raap A. K. , Tanke H. J. 
Published: August 2005

AbstractThis study aims at the quantification of specific DNA sequences by using fluorescence in situ hybridization (ISH) and digital imaging microscopy. The cytochemical and cytometric aspects of a quantitative ISH procedure were investigated, using human peripheral blood lymphocyte interphase nuclei and probes detecting high copy munber target sequences as a model system. These chromosome‐specific probes were labeled with biotin, digogigenin, or fluoresceinQuantification of the fluorescence ISH signals was performed using an epifluorescence microscope equipped with a multi‐wavelength illuminator, and a cooled charge coupled device (CCD) camera. Specific image analysis programs were developed for the segmentation and analysis of the images provided by ISHThe fluorescence intensity distributions of the ISH spots showed large internuclear variation (CVs up to 65%) for the probes used. The variation in intensity was found to be independent of the probe, the type of labeling, and the type of immunocytochemical detection used. Variation in intensity was not caused primarily by the immunocytochemical detection method, since directly fluorescein‐labeled probes showed similar internuclear variation. Furthermore, it was found that different white blood cell types, which harbor different degrees of compactness of the nuclear chromatin, showed the same variationThe infra‐nuclear variation in intensity of the ISH spots on the two chromosome homologs within one nucleus was significantly smaller (approximately 20%) than the inter‐nuclear variation, probably due to more constant local hybridization conditions. Due to the relatively small intranuclear variation, copy number polymorphisms of the satellite DNA sequence on chromosome 1 could readily be quantified. Furthermore, an internal standard to correct for differences in hybridization efficiency among cells, based on a second hybridization, was introducedIt is concluded that development of an accurate quantitative ISH procedure requires further reduction of the internuclear variation. Results presented indicate that improvements in the preparation of the target material and in the hybridization reaction are especially needed to fulfill this requirement© 1992 Wiley‐Liss, Inc.
Flow cytometric determination of atypical antigen expression in acute leukemia for the study of minimal residual disease
Authors: Drach Johannes , Drach Doris , Glassl Herta , Gattringer Claus , Huber Heinz 
Published: August 2005

AbstractThe aim of this study was to investigate to which extent acute leukemias could be monitored for residual disease by using atypical antigen combinations as leukemia‐related markers. Atypical antigenic features were determined by double color flow cytometry and included coexpression of lymphoid and myeloid related antigens, unnphysiological coexpression of immature and mature antigens, and lack of an antigen that is normally expressed during maturation. Atypical immunophenotypes were detected in 35 of 68 patients with AML (51.5%) and 15 of 24 patients with ALL (62.5%). When 12 patients with leukemia‐associated markers were again analyzed at relapse, the relevant antigen combinations were retained in 11 of them. The sensitivity of this two color flow cytometric assay as determined in dilution experiments was 1 in 103 to 104 cellsFollow‐up studies of bone marrow samples revealed that, after induction chemotherapy cells with leukemia‐associated markers were detectable in several patients at a frequency of 0.5 to 4%, but only patients in whom the cells with atypical antigens never disappeared suffered from relapse. In contrast, patients who became negative for the atypical cells remained in complete remission (median remission duration after the first negative bone marrow assessment by flow cytometry 52 weeks, range 20–102)We conclude that atypical antigen combinations, which are present in a meaningful number of acute leukemias, are a valuable means of monitoring acute leukemia patients during follow‐up. This flow cytometric approach can complement other strategies to get a more accurate definition of remission in acute leukemia. © 1992 Wiley‐Liss, Inc.
Flow cytometric analysis of altered mononuclear cell transmembrane potential induced by cyclosporin
Authors: Tanner Michael K. , Wellhausen Samuel R. , Klein Jon B. 
Published: August 2005

AbstractImmunosuppression by the fungal metabolite cyclosporin A (CsA) is characterized by functional inhibition, rather than destruction of cells. Because activation of immune cells involves intracellular signalling events associated with modulations of cell transmembrane potential (TMP), we tested the ability of cyclosporin A (CsA) to modulate immune mononuclear cell TMP in vitro using a TMP sensitive cationic dye, dihexyloxacarbocyanine (DIOC6(3)). All analyses were performed by flow cytometry. CsA increased TMP in monocytes and lymphocytes isolated from the blood of healthy human volunteers. CsA‐induced hyperpolarization was time and concentration dependent in monocytes while the lymphocyte hyperpolarization, although time dependent, was evident over the entire range of CsA concentrations tested. CsA‐induced hyperpolarization of lymphocytes was dependent on potassium ion (K+) efflux as indicated by the absence of hyperpolarization in 154 mM KCl or with pretreatment with 100 üMM quinine (an inhibitor of K+ channels). Monocyte hyperpolarization by CsA was not inhibited in either system. Dihydrocyclosporin C (DH‐CsC), an immunosuppressive analog of CsA, also hyperpolarized mononuclear cells. The anionic TMP sensitive dye bis oxonol (di‐BA‐C4) indicated that CsA treatment depolarized mononuclear cell plasma membranes. The mitochondrial poison carbonyl cyanide p‐trifluoromethoxy‐phenyl‐hydrazine (FCCP) eliminated CsA induced hyperpolarization and also indicated that CsA caused plasma membrane depolarization. We conclude that brief in vitro exposure to cyclosporin alters the transmembrane electrical potential of human lymphocytes and monocytes. © 1993 Wiley‐Liss, Inc.
Micromanipulation by “multiple” optical traps created by a single fast scanning trap integrated with the bilateral confocal scanning laser microscope
Authors: Visscher K. , Brakenhoff G. J. , Krol J. J. 
Published: August 2005

AbstractWe have developed a novel micromanipulator consisting of multiple optical traps created by scanning one single beam trap along a variable number of positions. Among other things, this enables the orientation of irregularly shaped and relatively large structures which could not be oriented by just one trap as is demonstrated on long Escherichia coli bacteria filaments. We expect that the multiple trap manipulator will broaden the field of applications of optical trapping as a micromanipulation technique. For example, it facilitates the study of mechanical properties of extended structures as illustrated by a “bending”‐experiment using E. coli bacterium filaments. A special application of the multiple trap manipulator is the “indirect trapping” of objects which we did by keeping them held between other optically trapped particles. Indirect trapping makes it possible to trap particles which either cannot be trapped directly due to their optical properties (refractive index) or for which exposure to the laser radiation is undesirable. The multiple optical trap manipulator is controlled interactively by a UNIX workstation coupled to a VME instrumentation bus. This provides great flexibility in the control of the position and the orientation of the optical traps.Micromanipulation makes it desirable to have real time 3D microscopy for imaging and guidance of the optical traps. Therefore we integrated optical micromanipulation and a specially developed real‐time confocal microscope. This so called bilateral confocal scanning laser microscope (bilateral CSLM) [Brakenhoff and Visscher, J Microsc 165:139–146, 1992] produces images at video rate. © 1993 Wiley‐Liss, Inc.
Optimizing fluorescent in situ hybridization with rRNA‐targeted oligonucleotide probes for flow cytometric identification of microorganisms
Authors: Wallner Günter , Amann Rudolf , Beisker Wolfgang 
Published: August 2005

AbstractA combination of fluorescent rRNA‐targeted oligonucleotide probes (“phylogenetic stains”) and flow cytometry was used for a high resolution automated analysis of mixed microbial populations. Fixed cells of bacteria and yeasts were hybridized in suspension with fluorescein‐ or tetramethylrhodamine‐labeled oligonucleotide probes complementary to group‐specific regions of the 16S ribosomal RNA (rRNA) molecules. Quantifying probe‐conferred cell fluorescence by flow cytometry, we could discriminate between target and nontarget cell populations. We critically examined changes of the hybridization conditions, kinetics of the hybridization, and posthybridization treatments. Intermediate probe concentrations, addition of detergent to the hybridization buffer, and a posthybridization washing step were found to increase the signal to noise ratio. We could demonstrate a linear correlation between growth rate and probe‐conferred fluorescence of Escherichia coli and Pseudomonas cepacia cells. Oligonucleotides labeled with multiple fluorochromes showed elevated levels of non‐specific binding and therefore could not be used to lower the detection limits, which still restrict studies with fluorescing rRNA‐targeted oligonucleotide probes to well‐growing microbial cells. Two probes of different specificities—one labeled with fluorescein, the other with tetramethylrhodamine—could be applied simultaneously for dual color analysis. © 1993 Wiley‐Liss, Inc.