bluuebunny/crossref_metadata_2025_split huggingface dataset:
Analytical methods for the study of liver cell proliferation
Authors: Gerlyng Per , Stokke Trond , Huitfeldt Henrik S. , Stenersen Tom , Danielsen Håvard E. , Grotmol Tom , Seglen Per O. 
Published: August 2005

AbstractVarious cytometric methods for analysis of regenerating rat liver growth (DNA ploidy distributions, binucleation, and DNA synthesis by in vivo BrdUrd incorporation) were evaluated. The overall hepatocellular growth rate (labeling index), the binucleation rate, and separate indices for mononuclear and binuclear cells could be measured simply by microscope counting of collagenase‐isolated hepatocytes immunostained for BrdUrd. Flow cytometry of cells stained for BrdUrd and DNA provided labeling indices for the various hepatocellular DNA ploidy classes as well as for nonparenchymal cells (identified by their size‐dependent light scatter), but could not distinguish between mononuclear and binuclear hepatocytes. Image cytometry, using fluorescence or Feulgen staining, was inferior to flow cytometry in terms of speed and DNA resolution, but allowed a complete analysis of all hepatocellular DNA ploidy and nuclearity classes. It may therefore be the method of choice, particularly for analysis of liver cell cultures from which single cells are not easily obtained. Fluorescence staining would seem to be preferable to Feulgen staining, since the latter could not be used simultaneously with BrdUrd staining and therefore required a two‐step analysis. A non‐immunological method, based on the ability of incorporated BrdUrd to quench DNA staining by a Hoechst dye, could only be applied to isolated nuclei, thus giving no information about binucleation. The latter method may be useful for analysis of tumors which are difficult to dissociate to intact whole cells.
A statistical analysis of flow cytometric determinations of phagocytosis rates
Authors: Fredrickson A. G. , Hatzis Christos , Srienc Friedrich 
Published: August 2005

AbstractUptake of particles by phagocytosing cells is a process that exhibits variability of its rate. This variability is inherent in the mechanism of particle uptake and in the mechanisms that determine the distribution of physiological states within a population of phagocytosing cells. When numbers of particles ingested by cells are determined flow cytometrically an additional measurement variability is superimposed on and interacts with the aforementioned biological variability. In one method of determining population phagocytosis parameters, which involves fitting theoretical equations to experimental time course data on the fractions of cells which have ingested 0, 1, 2, particles, the effects of measurement variability are circumvented, although this usually has the cost of not using all the sample data obtained. However, in a second, simpler, method which is based on determining the time course of the number of particles ingested by an average cell, measurement variability is not circumvented and its effects must be considered. An analysis of the combined effects of biological and measurement variability on the results obtained with the simpler method is presented in this paper. Experimental results for phagocytosis of latex microspheres of uniform size and fluorochrome content by populations of the ciliate Tetrahymena pyriformis show that, for this system, measurement variability is entirely negligible in comparison with biological variability. This conclusion might not apply to other systems, however, and situations which might make measurement variability of some significance are mentioned in the paper. The equations given can be used for the analysis of such situations.
Clinical Applications of Cytometry: 6th Annual Meeting
Authors: Giorgi J. V. , Hurtubise P. E. , Cram L. S. , Parker J. W. , La Via M. F. 
Published: August 2005

AbstractThe Sixth Annual Clinical Applications of Cytometry Meeting was held September 11–14, 1991, in Charleston, SC. Attendance reached a record 470. The meeting provides a forum for interactions among investigators who utilize cytometry as a tool in their clinical immunology, cell biology, hematology, and cancer investigations. Clinical laboratory directors and their technical staff find the meeting of practical value because of the presentation of new applications that they can take home to their own laboratories. The emphasis of the meeting is on advances in the application of cytometry to clinical problems. Often, advances result from new dyes or reagents or improved instrumentation. Sometimes they result from advances in biology that make the studies possible. Occasionally a new way of looking at the same data provides a useful answer. In every case, the effort is to provide a reliable, straightforward way to quantitate biologic information in order to provide improved diagnosis or treatment of human disease.
Quantitation and mapping of integrated human papillomavirus on human metaphase chromosomes using a fluorescence microscope imaging system
Authors: Callahan Daniel E. , Karim Amna , Zheng Gemin , Ts,o Paul O. P. , Lesko Stephen A. 
Published: August 2005

AbstractIntegrated human papillomavirus type 16 (HPV‐16) DNA was directly visualized on metaphase chromosomes in the two human cervical carcinoma cell lines SiHa and CaSki by fluorescence in situ hybridization with a biotinylated DNA probe (7.9 kb). The fluorescence intensities of hybridization signals from single copies and dispersed clusters of integrated HPV‐16 DNA were quantified using a microscope equipped with a cooled‐CCD camera that was interfaced to an image processor and host computer. Hybridization signals were localized on chromosomes using separate, registered images of 4′, 6‐diamidino‐2‐phenylindole (DAPI) or propidium iodide stained metaphase chromosome spreads. In both SiHa and CaSki spreads, a single fluorescein signal was observed on one or both chromatids of chromosome 13, which was identified by simultaneous hybridization with a biotinylated centromere probe specific for chromosomes 13 and 21. Ratios of the distance from 13pter to the HPV‐16 signals to the entire chromosome length were 0.63 ± 0.05 in both SiHa and CaSki cells, indicating the possibility of a common integration domain on chromosome 13. In SiHa cells, no additional signals were observed on other chromosomes. This observation, taken together with literature reports that SiHa cells contain 1 to 2 copies of the HPV‐16 genome in this region of chromosome 13, suggests that each fluorescein signal on chromosome 13 represents one equivalent of the HPV‐16 genome. The total integrated fluorescence intensity in isolated CaSki metaphase chromosome spreads was approximately two orders of magnitude greater than that of a single copy of HPV‐16 DNA in SiHa cells, indicating an increase in HPV‐16 copy number. The relative increase in copy number indicated was similar to that reported in the literature.
Immunofluorescent labeling of centromeres for flow cytometric analysis
Authors: Schmitz Annette , Guilly Marie‐Noëlle , And Barbara Trask , Engh Ger Van Den 
Published: August 2005

AbstractA procedure to stain the centromeric region of chromosomes for dual beam flow cytometric analysis is described. Serum from a CREST (Scleroderma syndrome) patient presenting a high titer of anticentromeric antibodies was chosen on the basis of specificity of labeling of cells on slides. The high affinity of the antibodies to centromeres and low binding to chromosomal arms allowed the development of an indirect immunofluorescent labeling procedure using isolated and unfixed chromosomes stabilized by Mg++ ions. Discontinuous Ficoll gradients were used to separate chromosomes from unbound antibodies. With this procedure, chromosome clumping and degradation were minimal. The chromosomes were then stained with the DNA dyes Hoechst 33258 and chromomycin A3, before dual beam flow cytometric analysis. Flow karyotypes, with good chromosome peak resolution, were obtained for both human and hamster chromosomes subjected to the immunolabeling procedure. For quantification of FITC fluorescence due to bound antibody, chromosomes were counterstained with Hoechst only. The FITC intensity of antibody‐labeled human and hamster chromosomes were 4–10 and 20 times greater than control chromosomes, respectively. These results suggest that the staining procedure may be suitable for immunolabeling of chromosomes with antibodies recognizing other nuclear proteins and their subsequent quantification by flow cytometry.
A quantitative method for evaluating bivarlate flow cytometric data obtained using monoclonal antibodies to bromodeoxyuridine
Authors: Allen White R. , Terry Nicholas H. A. 
Published: August 2005

AbstractA method is presented for analyzing data from bivariate analysis of cell populations exposed to bromodeoxyuridine and subsequently examined both for the presence of BrdUrd and for the cellular DNA content. It is shown that certain features may be defined in the bivariate data which are constant independent both of cell type and, within limits, experimental variability. These landmark features include the ratio of red, DNA, fluorescence of G2 + M cells to G1 cells, the ratio of green fluorescence corresponding to the non‐specific binding of unlabeled G2 + M cells to unlabeled G1 cells, and the distribution of green fluorescence in unlabeled cells. The landmarks make it possible to standardize rules for establishing the separation line between‐labeled and unlabeled cells as required in these experiments to obtain estimates of cytokinetic parameters. Values obtained for the DNA synthesis time and the potential doubling time which result from different decision rules for distinguishing labeled from unlabeled are compared in two murine tumor lines. The potential doubling time, but not the DNA synthesis time is shown to depend sensitively on the separation line. Suggestions are presented for analyzing clinical data with this procedure.
Flow cytometric detection of the mitochondrial BCL‐2 protein in normal and neoplastic human lymphoid cells
Authors: Aiello Antonella , Delia Domenico , Borrello Maria Grazia , Biassoni Daniela , Giardini Roberto , Fontanella Enrico , Pezzella Francesco , Pulford Karen , Pierotti Marco , Porta Giuseppe Della 
Published: August 2005

AbstractThe bcl‐2 proto‐oncogene, rearranged and deregulated in B‐cell lymphomas bearing the t(14;18) translocation, encodes an inner mitochondrial membrane protein that blocks apoptotic cell death. We have developed a sensitive immunofluorescence assay for the single‐ and multicolor flow cytometric analysis of bcl‐2 protein in relation to other markers and cell cycle, based on a fixation‐permeation step of cells with paraformaldehyde and Triton X100 and the use of a bcl‐2 specific monoclonal antibody (MoAb). As an application of this method, we have examined the expression of bcl‐2 in normal and neoplastic lymphoid cells. We have found that > 80% of normal Tand B‐cells are bcl‐2 positive; following in vitro mitogen activation, the bcl‐2 reactivity decreased slightly in the former but markedly in latter cells. In both cases the bcl‐2 expression was not restricted to a specific phase of the cell cycle, as evidenced by two‐color analysis. On lymphoblastoid cell lines, the bcl‐2 staining intensity was variable and not necessarily correlated to molecular rearrangements of the bcl‐2 gene. Among fresh B‐cell non‐Hodgkin's lymphomas (B‐NHL), most sporadic Burkitt's cases were bcl‐2 negative. Of four centroblastic‐centrocytic cases with rearrangements of the bcl‐2 gene, only two presented elevated amounts of bcl‐2 protein, indicating that the levels of bcl‐2 are not diagnostic of the translocation. The flow cytometric analysis of bcl‐2 protein allows study and quantification, as the single cell level and in selected cell subsets, of the expression of the bcl‐2 gene and provides an important tool for assessing its role in hematopoietic cell development, proliferation, and neoplastic conversion.
Seric factors influence cell cycle modifications during commitment of murine erythroleukemia cells to differentiation
Authors: Mencherini Fabienne , Khochbin Saadi , Chabanas Agnès , Lawrence Jean‐Jacques 
Published: August 2005

AbstractCell cycle modifications are among the early events which take place during the induced differentiation of murine erythroleukemia (MEL) cells; a transient accumulation of the cells in the G1 phase of the cell cycle, followed by a re‐entry of the cells into a proliferation state, has been described. In order to characterize a putative role of serum in such variations, we have studied the modifications of the cell cycle parameters when cells were induced to differentiate in the presence or in the absence of seric factors. We show that, in the absence of exogenous factors brought by serum, the G1 accumulation was enhanced both in amplitude and in duration, but cells were still able to bypass the G1 block and re‐enter into the S phase. These results indicate that the resumption of cell proliferation after the transient block is under synergistic control of seric and endogenous factors, but these later are sufficient to overcome the block. However, MEL cells, were unable to differentiate in the absence of seric factors, as measured by the number of benzidine‐positive cells during induction with hexamethylene‐bisacetamide (HMBA) or butyric acid. This capacity to differentiate was recovered when serum was added back to the culture medium, and the efficiency of recovery was maximal when cells underwent a full round of DNA replication in the presence of serum after the G1 block. The analysis of two molecular markers of cell differentiation conned these results.
Dose effects of LPS on neutrophils‐ in a whole blood flow cytometric assay of phagocytosis and oxidative burst
Authors: Böhmer R. H. , Trinkle L. S. , Staneck J. L. 
Published: August 2005

AbstractWhole blood phagocytosis (P) and oxidative burst (OB), a rapid and sensitive flow cytometric method for quantifying neutrophil activation, was modified for single laser systems by using propidium iodide labeled Staphylococcus aureus and 2′,7′ dichlorofluorescein diacetate. The purpose of the present study was to characterize this assay with respect to the stimulatory activity of bacterial Upopolysaccharide (LPS) on phagocytosis. Blood from healthy donors was preincubated with log doses of bacterial LPS B (0. 1–1,000 ng/ml) or sterile pyrogen‐free saline at 37°C from 0–120 minutes. LPS increased both P and OB in a dose‐dependent manner (up to 62 and 121%, respectively) at all time points tested, and this effect on P and OB could be detected even with no preincubation. This LPS‐induced phagocytic activity could be blocked by the addition of polymyxin B (10 pμg/ml) during preincubation. The priming effect of LPS was maximal at 45 min. P and OB were inhibited by preincubation with EDTA, at doses > 2 mM (60 and 80% inhibition, respectively). These observations are consistent with the exquisite sensitivity of the neutrophil to endotoxin. This method can evaluate neutrophil response to immunomodulatory and chemotherapeutic agents in a physiological milieu. These findings re‐emphasize the necessity of using pyrogen‐free reagents in any study of neutrophil function.
On the measurement of cytokinetics by continuous labeling with bromodeoxyuridine with applications to chick wing buds
Authors: Allen White R. , Fallon John F. , Savage Mary P. 
Published: August 2005

AbstractThe cytokinetic properties, specifically the phase‐transit times, TG1, TS, and TG2+M, of chick wing bud cells were estimated using data obtained from continuous labeling of stage 20 embryos with bromodeoxyuridine (BrdUrd). The presence of BrdUrd was detected with monoclonal antibodies, and the amount of DNA in the cells was determined with propidium iodide. The fraction of cells in each cell cycle phase, the fraction of labeled cells, and the relative movement, a measure of the mean DNA content, of all labeled cells were evaluated using bivariate flow cytolnetry at successive times following introduction of the label. Equations are presented to describe the fraction of unlabeled cells in G2 + M; which gives a direct estimate of TG2 +M; the fraction of all labeled cells, whiA can then be used to estimate TG1; and, finally, the relative movement, which provides an estimate of TS. Thus, the data measured in these experiments together provide estimates of the progression through the cell cycle of limb mesoderm cells.
Eukaryotic DNA replication is a topographically ordered process
Authors: Humbert Catherine , Usson Yves 
Published: August 2005

AbstractThis paper describes the relationship between the BrdUrd replicating pattern of a cell and its localization within the S phase by means of topographical features and DNA content measurement. The present study follows an objective ranking of the, BrdUrd patterns obtained from a spectral analysis of the BrdUrd images. The pattern ranking was consistent with the DNA content increase throughout the S phase. Five texture groups were arbitrarily set up for the purpose of multivariate analysis. Nine topographical parameters were computed for each BrdUrd‐labelled nucleus. The descriptive quality of these parameters was assessed by means of factorial discriminant analysis. These parameters made it possible to characterize objectively the known pattern distributions of replication sites qualitatively described in the literature. © 1992 Wiley‐Liss, Inc.
Differences between platelet and microparticle glycoprotein IIb/IIIa
Authors: Nomura Shosaku , Suzuki Masahiko , Kido Hirofumi , Yamaguchi Kazuyuki , Fukuroi Tsutomu , Yanabu Mutsumasa , Soga Tetsuji , Nagata Hirokazu , Kokawa Terutoshi , Yasunaga Kojiro 
Published: August 2005

AbstractGlycoprotein (GP) IIb and IIIa are major constituents of the platelet membrane which are involved in forming the fibrinogen receptor on activated platelets. We used flow cytometry to study the effects of ethylene‐diamine tetraacetic acid (EDTA) on the membrane GPIIb/IIIa complexes of platelets and microparticles, and to study the effects of cations on dissociated GP complexes. Microparticles were detected by both the volume signal and by fluorescence using an FITC‐conjugated anti‐GPIb antibody (NNKY5–5). When platelets were stimulated with ADP, calcium ionophore A23187, or thrombin, fibrinogen binding to the platelet surface increased markedly. However, fibrinogen binding to microparticles showed little increase in response to such agonists. Microparticle GPIIb/IIIa complexes were dissociated by incubation with EFTA at 37°C but did not reassociate after treatment with divalent cations (Ca2+, Mg2+, and Mn2+) in contrast to platelet GPIIb/IIIa complexes. These results suggest that some interaction of GPIIb/IIIa and linked structures like the platelet cytoskeleton may be involved in the reassociation of dissociated GPIIb and GPIIIa, perhaps explaining the failure of reassociation of microparticle GPIIb/IIIa (i.e., the fibrinogen binding to microparticles). © 1992 Wiley‐Liss, Inc.
Evaluation of a method for detection of cells with reduced drug retention in solid tumours
Authors: Carpentier Y. , Gorisse M. C. , Desoize B. 
Published: August 2005

AbstractA method for detection of cells with reduced drug retention was evaluated in solid tumours. After a 1 h incubation with daunorubicin (DNR), the right angle scatter (RAS), forward angle scatter (FAS), and specific fluorescence (Fluo) were measured in sensitive and resistant cells; only Fluo was related qualitatively, but not quantitatively, to resistance. Various incubation conditions were examined. When the pH of the incubation medium increased, the DNR retention increased in sensitive and resistant cells. In contrast, when the cell concentration increased, the DNR retention decreased. Using sensitive and resistant cell lines, a proportion of resistant cells lower than 10% can be detected in a mixture. To analyse cells from solid tumours, the cells were dissociated by repeated fine needle aspirations. Tumours from 22 patients have been processed with this technique; 8 samples were classified as S (sensitive); 2 as R (resistant); and 12 as I (intermediate).Further experiments were run to study and improve the method. Another method of detection of dead cells was tested. The intra‐assay variability of the technique was found to be less than 10%. When the study was performed with different fragments of the same tumour, the variation, corresponding to the tumour heterogeneity, rose to 21 to 36%. The inter‐assay reproducibility was too high, so a variant of this technique has been adapted, using verapamil or cyclosporin A, which is able to block DNR efflux; this new method allows tumour cells to be used as their own controls. © 1992 Wiley‐Liss, Inc.
Cell cycle analysis of asynchronous cell populations by flow cytometry using bromodeoxyuridine label and Hoechst‐propidium iodide stain
Authors: Ormerod Michael G. , Kubbies Manfred 
Published: August 2005

AbstractContinuous labelling of cells with deoxybromouridine (BrdUrd) followed by staining with a bis‐benzimidazole (Hoechst 33258) and a phenanthridinium (propidium iodide or ethidium bromide) allows the cells to be separated by flow cytometry according to the extent of their DNA replication. This BrdUrd‐Hoechst/PI method has been used mainly to observe perturbations of the cell cycle in synchronously growing cells. In this paper we demonstrate that, when the method is applied to asynchronously dividing cells, more extensive information can be derived about the effects of cytotoxic and other treatments on the kinetics of the cell cycle. The interpretation of the data is explained, the effects of different types of cytotoxic agent are described, and the method is compared briefly to other methods for following cell cycle kinetics. © 1992 Wiley‐Liss, Inc.
Flow cytometric measurement of lipid peroxidation in vital cells using parinaric acid
Authors: Hedley David , Chow Sue 
Published: August 2005

AbstractA method for measuring lipid peroxidation using time resolved flow cytometry is described. Because of its chemical nature, the naturally fluorescent fatty acid cis‐parinaric acid is readily consumed in lipid peroxidation reactions. It could be loaded into Chinese hamster ovary cells in a time and concentration dependent manner at 37°C, with 5 μM for 60′ giving consistent, bright fluorescence without evidence of cytotoxicity. Examination of cells by fluorescence microscopy showed diffuse staining of surface and internal membranes. Cells were maintained at 37°C while being examined in an Epics Elite flow cytometer equipped with a 325nm HeCd laser, and parinaric acid fluorescence at 405nm was measured over time. Addition of the oxidant tert‐butyl hydroperoxide resulted in a burst of intracellular oxidation, shown by simultaneously loading the cells mrith dichlorofluorescein, and loss of parinaric fluorescence over time. This was followed by cell death, indicated by loss of forward light scatter and uptake of propidium iodide.Pretreatment of the cells with the antioxidant α‐tocopherol, 200 μM, reduced the rate of loss of parinaric acid fluorescence and delayed the onset of cell death. Simultaneous biochemical determination of the lipid peroxidation breakdown product malondialdehyde confirmed a close temporal relationship with loss of parinaric acid fluorescence, both with and without α‐tocopherol pretreatment and suggested that the flow cytometric assay for lipid peroxidation is of comparable sensitivity.The mitochondrial stain dodecyl acridine orange and the cyanine dye DiOC(6)3 were combined with cis‐parinaric acid staining and could be excited by the latter using resonance energy transfer. Because these two probes show a degree of organelle specificity, they can be used to measure the loss of parinaric acid due to lipid peroxidation at defined subcellular sites. Although our own interest in the method is to examine the actions of redox cycling anticancer drugs and the integrity of host antioxidant defences as a possible mechanism of drug resistance, it appears to be a versatile technique for investigating an important process of cell injury that is difficult to study using standard biochemical assays. © 1992 Wiley‐Liss, Inc.