bluuebunny/crossref_metadata_2025_split huggingface dataset:
S‐phase arrest of nutrient deprived lung cancer cells
Authors: Tinnemans Monique M. F. J. , Lenders Marie‐Hélène J. H. , ten Velde Guul P. M. , Blijham Geert H. , Ramaekers Frans C. S. , Schutte Bert 
Published: June 2005

AbstractThe human small cell lung cancer cell line NCI‐H82 was used to study the effect of nutritional status on cell proliferative parameters. Incorporation of bromodeoxyuridine (BrdUrd) was used to characterize actively proliferating cells and to obtain information on cell cycle dynamics. During several days, in which the culture medium was not changed, a gradual decrease in overall cell growth, labeling index, and vitality was observed. Simultaneously, an increase in the number of S‐phase cells that did not incorporate BrdUrd was noticed. From a more detailed kinetic study on d 6 of nutrient depletion, it appeared that, although the cells incorporated BrdUrd, they stopped cycling. When the same cells were regrown in fresh culture medium, a delay of 10 h in G1‐phase entry and exit was measured. After this delay the cells resumed the cell cycle at normal phase transit rates. In addition, BrdUrd unlabeled S‐phase cells were gradually lost from the culture. Bivariate flow cytometric DNA/ proliferating cell nuclear antigen (PCNA) and DNA/ Ki67‐antigen analyses confirmed a delay in G1 phase entry and exit. In this paper we show that nutrient depletion can cause cell cycle arrest as indicated by the occurrence of BrdUrd unlabeled S‐phase cells. This arrest could lead to overestimation of kinetic parameters such as S‐phase transit time (Ts) and potential time (Tpot) as determined after in vivo labeling of tumors. © 1995 Wiley‐Liss, Inc.
Flow cytometric monitoring of allergen induced basophil activation
Authors: Gane Pierre , Pecquet Catherine , Crespeau Hervé , Lambin Patrick , Leynadier Francisque , Rouger Philippe 
Published: June 2005

AbstractHuman basophils express many clustered differentiation antigens (CD), including CD45; however, none are specific for these cells. In a previous study, we described a two‐color immunofluorescence procedure, employing antibodies to CD45 and IgE for the cytometric evaluation of basophils. In the present work, we show that when sensitized basophils are stimulated by allergenic preparations, they demonstrate an upregulation of CD45 as well as a decrease in anti‐IgE binding. Since CD45 antigen modulation was observed with all aeroallergens tested and the decrease in IgE expression varied with allergenic preparations, the measurement of CD45 upregulation was used to evaluate basophil activation. Using this approach, reproducible results were observed when atopic patients were tested at different time intervals. In addition, we show that the upregulation of CD45 on allergen stimulated basophils is a very rapid phenomenon that is observed after a few minutes and that this rapid flow cytometric method can also be used for investigations on nonallergenic mediators of basophil stimulation such as calcium ionophores. © 1995 Wiley‐Liss, Inc.
Differential effects of two fluorescent probes on macrophage migration as assessed by manual and automated methods
Authors: Denholm Elizabeth M. , Stankus Gerald P. 
Published: June 2005

AbstractFluorescent probes have been utilized to label leukocytes for both in vivo and in vitro studies of cell migration; however, the effects of such probes on migration have not been determined. The aim of this study was to examine the effects of two commonly used fluorescent probes on leukocyte chemotaxis. J774 macrophages were labeled with either calcein‐acetoxymethyl ester (calcein‐AM) or 2′,7′‐bis‐(2‐carboxyethyl)‐5‐(and‐6)‐carboxyfluorescein, acetomethyl ester (BCECF‐AM), then assayed for their ability to migrate to zymosan‐activated serum (ZAS). Cell migration was quantified by two methods: visual counting of cells and measuring cell fluorescence. Using the cell counts, commison of unlabeled and fluorescently labeled macrophages demonstrated that BCECF‐AM decreased the number of cells responding to ZAS, while cal‐n‐AM had essentially no effect. Neither probe significantly affected the number of cells migrating to medium alone. The inhibitory effects of BCECF‐AM on cell migration increased with probe concentration (0.1–1.0 μM) and cell fluorescence. Cell viability was unaffected by either probe. In contrast to the results obtained by visual counting, measuring fluorescence of migrated cells did not reveal a significant difference between the chemotactic response of macrophages labeled with BCECF‐AM and those labeled with calcein‐AM. These experiments indicated that fluorescent probes can affect the chemotactic response and that inhibitory activity of these probes may not be detected when chemotaxis is quantified solely by automated methods. © 1995 Wiley‐Liss, Inc.
Enumeration of viable Candida albicans blastospores using tetrabromofluorescein (Eosin Y) and flow cytometry
Authors: Costantino Paul J. , Budd Dianne E. , Gare Norman F. 
Published: June 2005

AbstractA rapid assay was developed for determination of the viability of blastospores of Candida albicans utilizing flow cytometry to detect the accumulation of tetrabromofluorescein in non‐viable yeast cells. Eosin Y was shown to stain non‐viable C. albicans blastospores selectively without affecting the cellular viability of competent yeast cells or resulting in non‐specific staining of viable cells. Results of these studies show that this flow cytometric method of determining cell viability of C. albicans is more accurate and more precise than the more common method of enumerating colony forming units. © 1995 Wiley‐Liss, Inc.
Organic material concentration in auditory outer hair cells measured by laser interferometry
Authors: Laffon E. , Dulon D. , Aurousseau C. , Dilhaire S. , Claeys W. 
Published: June 2005

AbstractOuter hair cells (OHC) of the mammalian cochlea are quasicylindrical cells of different length, which play a major role in hearing at threshold. Their particular shape allows the use of a noninvasive laser interferometric technique of isolated cells in vitro in order to measure the organic material concentration (OMC), hence the density of each cell body. In most (95 %) of the OHCs isolated from the same guinea pig, when the cell diameter is normalized, the results show that the cell body OMC does not vary with cell length. In different animals, the respective normalized OMC mean values can vary between 70 kg/m3 and 103 kg/m3. A few OHCs with morphological particularities often possess cell body OMCs > 103 kg/m3. The results of the interferometric measurements in isolated OHCs confirm that density variations in the cell bodies are not involved in a sound frequency coding. The in vitro OMC variations of the OHCs could be related to the isolation procedure; however, they could also correlate with actual in vivo OMC variations. © 1995 Wiley‐Liss, Inc.
Flow cytometric assay of lung macrophage uptake of environmental particulates
Authors: Stringer Bradley , Imrich Amy , Kobzik Lester 
Published: June 2005

AbstractWe sought to establish a quantitative method using flow cytometry to study uptake of environmental particulates by alveolar macrophages (AMs). We used right angle light scatter (RAS) to measure uptake of titanium dioxide, quartz, and diesel particulates. After incubation with TiO2 in vitro, AMs showed dose‐dependent increases in both cell‐associated particles visualized by microscopy and RAS measured by flow cytometry (e.g., fold increase RAS at 4, 8, 16, 32, and 80 μ/ml, respectively, = 2 ± 0.1, 4.0 ± 0.5, 5.5 ± 0.5, 9.1 ± 2.5, 14.3 ± 0.9; mean ± SEM). Similar results were obtained with quartz and diesel particles. A strong correlation was observed between particle load per cell and AM RAS after uptake of fluorescent latex beads or fluorescent TiO2 (coated with BODIPY‐BSA) (R2 = 0.984, 0.997, respectively). Using this technique, we found AM uptake of environmental particulates to be substantially greater than that of a panel of myelomonocytic and epithelial cell lines, consistent with their physiologic role in pulmonary defenses. RAS measurements have also identified both calcium‐dependent and calcium‐independent components in AM interactions with inert particles. Although this technique does not allow precise quantitation of particle number or mass per cell, flow cytometric analysis of relative increases in RAS is a useful tool to study AM interactions with a variety of environmental particulates. © 1995 Wiley‐Liss, Inc.
Karyotypic heterogeneity and its relation to labeling index in interphase breast tumor cells
Authors: Balazs Margit , Matsumura Kouji , Moore Dan , Pinkel Daniel , Gray Joe W. , Waldman Frederic M. 
Published: June 2005

AbstractWe have used fluorescence in situ hybridization (FISH) with chromosome‐specific probes and immunofluorescent detection of in vivo bromodeoxyuridine (BrdUrd) incorporation to evaluate simultaneously numerical chromosome aberrations and proliferative activity of breast cancers. The number of distinct hybridization domains specific for repetitive pericentromeric sequences on chromosomes 1, 7, 11, 15, 17, and X was used as an indicator of copy number of these chromosomes in interphase tumor cells from 23 human breast cancers. Every tumor analyzed showed a heterogencous distribution of copy number for at least one chromosome type. The copy number distribution for different chromosomes within a tumor frequently showed differing patterns. Major cell populations showing monosomy were relatively rare, occurring only in five cases for chromosome 17, once for chromosome 1, and once for chromosome 15. Flow cytometric analysis of DNA ploidy correlated well with FISH analysis, although flow cytometry failed to detect aneuploidy when only a few chromosomes were affected. To determine whether cell populations with different chromosomal copy numbers have identical proliferation characteristics in vivo, BrdUrd incorporation and centromeric copy number were detected simultaneously. Comparison of the chromosome copy number distribution in BrdUrd‐positive cells vs. the distribution of the entire cell population showed different distributions in seven of the 20 cases analyzed. This study demonstrates the common occurrence of chromosome copy number heterogeneity and suggests that a cell phenotype (proliferation) may be associated with genotypic subpopulations. © 1995 Wiley‐Liss, Inc.
Flow cytometric analysis using digital signal processing
Authors: Zilmer Nick A. , Godavarti Mahesh , Rodriguez Jeffrey J. , Yopp Timothy A. , Lambert Georgina M. , Galbraith David W. 
Published: June 2005

AbstractCurrent commercial flow cytometers employ analog circuits to produce the feature values of the pulse waveforms that result from particle analysis. The use of analog pulse processing limits the features that can be measured to pulse integral, pulse height, and pulse width, and a large amount of potentially relevant information about the shape of the pulse waveform is lost. Direct digitizing of the waveform provides a means for the extraction of additional features, for example, pulse skewness and kurtosis, as well as the Fourier properties of the pulse. Here we describe a digital pulse waveform processing system that is compatible both with a commercial flow cytometer, and with a readily available computational platform. The performance of the digital and analog systems were compared through analysis of synthetic waveforms, and the waveforms produced by standard fluorescence microspheres and biological particles. The digital waveform processing system was found to be accurate and flexible, and the value of several of its unique attributes was demonstrated using biological cells. A protocol was designed in which digital pulse processing provided a means for the quantitative monitoring of the optical alignment of the flow cytometer. It was shown that digital pulse processing could be used to discriminate between particle classes which produce feature values indistinguishable through analog pulse processing, and to discriminate accurately single cells from doublets and larger aggregates. © 1995 Wiley‐Liss, Inc.
Rapid flow cytometric method for measuring lymphocyte subset activation
Authors: Maino Vernon C. , Suni Maria A. , Ruitenberg Joyce J. 
Published: June 2005

AbstractStandard in vitro methods for assessing T‐cell activation have typically measured either the proliferative responses of peripheral blood mononuclear cell (PBMC) cultures to various provocative stimuli employing tritiated thymidine incorporation or the secretion of specific cytokines from activated cells. These bulk assay methods suffer the drawback of being lengthy assays, and, in addition, they do not provide information about functional responses of individual lymphocyte subsets. This report describes a three‐color flow cytometric method for the rapid analysis (4 hours) of individual T‐cell subsets in whole blood responding to various provocative stimuli, including pokeweed mitogen, the comitogenic monoclonal antibodies (mAbs) CD2/CD2R, the superantigen staphylococcal enterotoxin B (SEB), and the specific antigen Candida albicans. After 4 hours, CD69 expression in response to CD2/CD2R paralleled thymidine incorporation measured after 72 hours. Variations in the proportions of CD4+ and CD4− T cells expressing CD69 were observed with different stimuli. These observations demonstrate the potential of multiparameter flow cytometry for the investigation of functional responses of individual T‐cell subsets to a variety of stimuli in whole blood. © 1995 Wiley‐Liss, Inc.
Cell cycle progression and phenotypic modification of Ki67 antigen‐negative G1‐ and G2‐phase cells in phorbol ester‐treated molt‐4 human leukemia cells
Authors: Tsurusawa Masahito , Fujimoto Takeo 
Published: June 2005

AbstractTo elucidate the relationship between the level of cellular Ki67‐reactive antigen and cell proliferation, the effects of 12‐O‐tetra‐decanoylphorbol 13‐acetate (TPA) on Ki67 expression, cell cycle progression, and surface phenotypes of human T‐lymphoblastic leukemia Molt‐4 cells were investigated by multiparameter flow cytometry. The Ki67 antigen is constitutionally expressed in almost all untreated exponentially proliferating Molt‐4 cells. Treatment with 10 nM TPA prolonged the duration of the cell cycle time and resulted in a progression arrest of cells in G1‐ and G2‐phases, during which Ki67 expression was decreased to an undetectable level. However, in TPA‐treated cultures, the Ki67‐positive fraction was invariably smaller than the growth fraction as estimated from continuous 5‐bromodeoxyuridine (BrdUrd) labeling curves. This discrepancy could be explained by the finding that some Ki67‐negative G1 cells do not enter the resting state but instead remain in the cycling compartment. These results show that Ki67 expression of tumor cells with relatively long G1 duration is downregulated to undetectable levels in late G1‐phase and the difference in the level of Ki67 expression between late G1 cells and resting G1 cells is undetectable by conventional immunological methods. Although TPA induced differentiation of Molt‐4 cells into mature suppressor T cells, the phenotypic modification was not correlated with cell cycle position and Ki67 reactivity of the cells. These results suggest that growth arrest and phenotypic differentiation of Molt‐4 cells are independent effects of TPA. © 1995 Wiley‐Liss, Inc.
A new flow cytometric method for discrimination of apoptotic cells and detection of their cell cycle specificity through staining of F‐actin and DNA
Authors: Endresen Petter C. , Prytz Per S. , Aarbakke Jarle 
Published: June 2005

AbstractDrug‐initiated apoptosis of human leukemia HL‐60, THP‐1, and U‐937 cells was studied via multiparameter flow cytometry and cell sorting. A new flow cytometric method that allows both identification and quantitation of apoptotic cells and estimation of their cell cycle specificity is presented. The method is based on paraformaldehyde fixation followed by staining of F‐actin and DNA with fluorescein isothiocyanate (FITC)‐phalloidin and propidium iodide (PI), respectively. Bivariate green fluorescence (F‐actin) vs. side scatterplots of HL‐60 cells treated with 10 μM etoposide for 4 h showed two cell populations, one with high green fluorescence and low side scatter and one with low green fluorescence and high side scatter. Sorting revealed cells with intact nuclei in the high green fluorescence/low side scatter population and cells with fragmented nuclei in the low green fluorescence/high side scatter population, demonstrating that the cells in the latter population were apoptotic. Exposure of HL‐60 cells to 10 μJM etoposide for 4 h resulted in S‐phase selective apoptosis, whereas 5 μg/ml cycloheximide initiated apoptosis mainly in G0/G1‐phase and S‐phase cells. The apoptotic response of HL‐60 cells to 20 GY γ‐irradiation was selective for S‐phase and G2 + M‐phase cells. The present method offers the opportunity to estimate the cell cycle distributions of both the apoptotic and the nonapoptotic cell populations, which is especially valuable when apoptosis occurs in association with cell cycle perturbations. A similar shift from one to two cell populations in green fluorescence vs. side scatterplots, similar to that observed for HL‐60 cells, was observed in the THP‐1 and U‐937 cell lines secondary to etoposide treatment. © 1995 Wiley‐Liss, Inc.
Single‐step procedure for labeling DNA strand breaks with fluorescein‐ or Bodipy‐conjugated deoxynucleotides: Detection of apoptosis and bromodeoxyuridine incorporation
Authors: Li Xun , Traganos Frank , Melamed Myron R. , Darzynkiewicz Zbigniew 
Published: June 2005

AbstractThe methods of in situ labeling of DNA strand breaks have been used to identify apoptotic cells and/or DNA replicating cells. While discrimination of apoptotic cells is based on the inherent presence of numerous DNA strand breaks in their chromatin, DNA proliferating cells can be discriminated by the selective DNA strand break induction by photolysis (SBIP) methodology at the sites that contain incorporated bromodeoxyuridine (BrdUrd) or iododeoxyuridine (IdUrd). In both instances, DNA strand breaks are labeled with biotin‐ or digoxygenin‐conjugated deoxynucleotides, preferably in the reaction catalyzed by exogenous terminal deoxynucleotidyl transferase; fluorescein tagged avidin (streptavidin) or digoxygenin antibody is used in the second step of the reaction. In the present study, DNA strand break labeling was simplified by using directly labeled deoxynucleotides, in a single‐step reaction. Cell fluorescence was measured by flow cytometry as well as by a microscope‐based laser scanning multiparameter cytometer. Apoptotic cells in HL‐60 cultures treated with camptothecin or in primary cultures of non‐Hodgkin's lymphoma cells treated with prednisolone were easily identified utilizing BODIPY‐conjugated dUTP (B‐dUTP). Apoptotic cells were also recognized using fluorescein‐conjugated dUTP or dATP, although the discrimination was more pronounced with B‐dUTP The single‐step procedure, requiring fewer centrifugation. steps, resulted in less cell loss compared to the two‐step cell labeling technique. The cells, labeled with BrdUrd for 30 min or 1 h, could also be identified in a single‐step reaction; under the present conditions, better discrimination (signal‐to‐noise ratio) was obtained measuring cell fluorescence with the microscope cytometer compared to cell analysis by flow cytometery. Preincubation of cells with Hoechst 33258 prior to ultraviolet (UV) illumination, to enhance the photolysis, improved detection of cells which incorporated BrdUrd. The morphology of cells subjected to SBIP was excellent, allowing visualization of distinct DNA replication points. Becaus, unlike the immunocytochemical methods used to detect BrdUrd incorporation, the SBIP methodology does not require DNA denaturation by heat or acid, nuclear proteins are expected to remain undenatured in situ, allowing one to study colocalization of various constituents, detected immunocytochemically, at the DNA replication points. © 1995 Wiley‐Liss, Inc.
Binding diversity of antibodies against external and internal epitopes of the multidrug resistance gene product P‐glycoprotein
Authors: Lehne G. , De Angelis P. , Clausen O. P. F. , Egeland T. , Tsuruo T. , Rugstad H. E. 
Published: June 2005

AbstractP‐glycoprotein (Pgp) is a trans‐membraneous protein that is associated with multidrug resistance (MDR) in human cancer, including hepatocellular carcinomas and leukemias. There is no consensus regarding methods of choice for analysis of Pgp expression, and development of reliable analytical methods is now essential. We have studied the Pgp expression in human hepatoma and leukemia cell lines using flow cytometry. The aim of the study was to compare binding properties of anti‐Pgp antibodies reacting with surface (MRK16, UIC2) and cytoplasmic (C219, JSB‐1) epitopes to assess which antibody performed best with respect to fluorescence discrimination. By histogram subtraction the fractions of resistant human hepatoma cells positive for Pgp were 99% (MRK16), 97% (UIC2), 77% (JSB‐1), and 51% (C219), demonstrating variations in antibody reactivity. The resolution in detecting decreasing levels of Pgp in hepatoma cells was superior for the externally binding antibodies, showing that there is a correlation between antibody reactivity and fluorescence discrimination. Similar results were obtained for parental and resistant KG1a human leukemia cell lines. The Pgp epitopes remained reactive to the anti‐Pgp MAbs after methanol fixation and cryopreservation. By dual parameter flow cytometry it was shown that Pgp expression in viable cells may be assessed together with uptake of epirubicin, which was low in cells expressing high levels of Pgp and vice versa. In conclusion, all tested antibodies proved useful for flow cytometric detection of high levels of Pgp, but the externally binding ones were superior in detection of low and variable levels of Pgp. © 1995 Wiley‐Liss, Inc.
Scatchard analysis of fluorescent concanavalin a binding to lymphocytes
Authors: Gordon Ian L. 
Published: June 2005

AbstractStandard Scatchard analysis of ligand binding to cell receptors requires the use of isotopes and is imprecise at low ligand concentrations. To evaluate the feasibility of Scatchard analysis via fluorescence flow cytometry, the binding of fluorescein isothiocyanate‐derivatized concanavalin A (FITC‐ConA) to murine lymphocytes at 4°C was compared to 125I‐ConA binding. A FACS IV flow cytometer (Becton‐Dickinson, Mountain View, CA) was used for analysis of cells after fluorescent ligand binding. A simple spectrophotometric technique was used to calibrate the relation between cytometer‐determined fluorescence and ligand binding per cell. As FITC‐ConA binding showed a quasi‐Gaussian distribution, the mean number of molecules bound per cell was easily calculated. Scatchard analysis of FITC‐ConA binding yielded results (1.9 × 106 receptors/cell, K = 3.6 × 10−15) similar to those obtained with 125I‐ConA (1.4 × 106 receptors/cell, K = 5.2 × 10−15). Cytometric Scatchard plots showed less scatter and seemed more precise, suggesting superiority to radioactive ligand measurements, particularly at low ligand concentrations. © 1995 Wiley‐Liss, Inc.