bluuebunny/crossref_metadata_2025_split huggingface dataset:
Flow cytometric analysis of small DNA content differences in heterogeneous cell populations: Human amniotic fluid cells
Authors: Koch H. , Bettecken T. , Kubbies M. , Salk D. , Smith J. W. , Rabinovitch P. S. 
Published: August 2005

AbstractAccurate flow cytometric determination of nuclear DNA fluorescence has been attempted with human amniocentesis materials employing a detergent nuclear isolation protocol and 4,6‐diamidino‐2‐phenylindole (DAPI) staining. Assay of native fluids yielded inadequate fluorescence histograms, whereas analysis of cultured amniocytes yielded unimodal histograms in 80 of 83 samples. Three fluids displayed unexpected bimodality, which may have resulted from the interaction between detergent and chromatin in rare cell type. Using a standardized protocol for preparation, staining, and assay, including brief periods of cocultivation of a human triploid reference standard, the percent experimental error in the flow cytometric DNA content estimate was as low as 0.2% in repeated assays of a single sample, and 0.4% in assays of a given genotype at different occasions. In two series of blindly assayed specimens, the percent standard error of the DNA content estimate ranged from 0.6 to 1.0%.
Correlation between protein synthesis rate and nuclear morphology of human erythroblasts as determined by quantitative autoradiography and high‐resolution image analysis
Authors: Dörmer P. , Abmayr W. , Giaretti W. 
Published: August 2005

AbstractHuman erythropoiesis is an appropriate model for analyzing correlations between functional activity of cells during maturation and nuclear morphology as determined by high‐resolution image analysis. In a previous study a relationship had been found between nuclear condensation and the proliferative activity of the cells. This present study analyzed the correlation between nuclear morphology and the protein synthesis rate. The latter was assayed by measuring the 14C‐I‐leucine incorporation rate utilizing the technique of quantitative 14C‐autoradiography. The labeled cells were first classified according to conventional cytological criteria into four groups of increasing maturity. Following grain counting the nuclei were Feulgen‐stained, and after removel of the silver grains, the local nuclear optical densities were evaluated by scanning.There was a nonlinear relationship between the protein synthesis rate and features representing the degree of chromatin condensation. This nonlinearity was explained by the mediator function of RNA, predominantly mRNA. The amount of protein produced at a given time depends on the transcriptional activity of the chromatin, the frequency of mitotic divisions partitioning the mRNA, and the half‐life of the mRNA. It was concluded that the chromatin texture of erythroblasts reflects three different metabolic activities: the rate of DNA strand duplication, the transcriptional activity for structural proteins enabling a cell to grow and cycle, and for functional proteins, particularly hemoglobin. Since these three activities appear to be synchronized, it is understandable that functional as well as textural features can be used to perform a supervised classification by means of multivariate analysis. Application of the five most significant features allowed a consistent classification of the erythroblasts into the various cytological compartments in 76.7% of all cases, while the classification error of the observer amounted to 13.7%.
Rare‐event analysis methods for detection of fetal red blood cells in maternal blood
Authors: Cupp James E. , Leary James F. , Cernichiari Elsa , Wood James C. S. , Doherty Richard A. 
Published: August 2005

AbstractMethods for analyzing very small subpopulations of cells (“rare‐event analysis methods”) are of interest in many areas of biology. In this paper we demonstrate the ability to detect one Rh‐positive erythrocyte per 100,000 Rh‐negative erythrocytes. This is the level needed to detect human fetal red blood cells in the maternal circulation in the case of fetal‐maternal Rh‐incompatibility.Erythrocytes stained with fluorescent immunobeads are more than 100 times as bright as the same cells stained by indirect immunofluorescence (or almost 40 times as bright as cells stained by the biotin‐avidin technique). More importantly there is greater than a sevenfold increase in signal‐to‐noise (S/N) ratio, which proves critical in detecting very low frequency („rare event”︁) cell subpopulations. Although equal mixtures of positive and negative cells can easily be detected, positives cannot be easily distinguished from negatives when they are present in relative frequencies of 1:1000 or less as measured by either indirect immunofluorescence or biotin‐fluorescent avidin labeling of D antigens. However, use of fluorescent immunobeads and high‐speed flow cytometry allows detection of subpopulations of the same cells at relative frequencies of less than 1:100,000. Based on calibrations using free‐fluorescent immunobeads, the average Rh‐positive cell (when present in a relative frequency of 1:100,000) labeled with 36 ± 19 fluorescent immunobeads. Rh‐positive cells of relative frequencies 1: 10,000 and 1: 100,000 can be detected above background (Rh‐negative red blood cells treated with the same amount of fluorescent immunobeads). Using defined mixtures of Rh‐positive and negative cells, the expected numbers of positive cells were observed over a range of 1:100 to 1:100,000 relative Rh‐positive cell frequencies. These methods should prove of general use in applications where original frequency information about rare cell subpopulations is important and would be lost if enrichment procedures were to be used.
The measurement of specific cell: Cell interactions by dual‐parameter flow cytometry
Authors: Segal David M. , Stephany David A. 
Published: August 2005

AbstractThe Fc receptor‐mediated aggregation of antibody‐coated spleen cells from the P388D1 mouse macrophage line was followed using a novel flow cytometric technique. P388D1 and spleen cells were directly labeled with green‐emitting (fluorescein isothiocyanate) and red‐emitting (substituted rhodamine isothiocyanate) flurophores, respectively. They were mixed, incubated in suspension at 4°C, and analyzed for aggregation with a dual laser flow cytometer. Unconjugated cells appeared as particles which were either red or green, while conjugates were detected as particles which were both red and green. Using this assay procedure, 5 × 104 cells were analyzed in 2‐3 min for the percentages of conjugates, free spleen cells, and free P388D1 cells. Intercellular aggregation required both antibody on the spleen cells and free Fc receptors on the P388D1 cells; nonspecific aggregates accounted for 1% or less of the total particles analyzed. Measurements of the fluorescence distributions within conjugates indicated that the majority of conjugates contained a single P388D1 cell bound to 1–3 spleen cells, and that only heterophilic aggregation occurred. The flow cytometric technique described here should be applicable for the measurement of the initial events of intercellular aggregation in other systems as well.
Cell sorting and microchemistry of cultured human fibroblasts: Applications in genetics and aging research
Authors: Jongkind Johan F. , Verkerk Anton 
Published: August 2005

AbstractCell sorting is a way to isolate viable subpopulations of cells present in a mixture. The drawback of the isolation method is the shortage of material for subsequent biochemical determinations. We have employed a combination of (ultra‐) microchemistry and cell sorting to overcome this problem. The methods enable determinations of protein and several enzyme activities on triton extracts of 5,000–10,000 sorted cells. In addition, using ultramicromethods we could determine enzyme activity in single sorted cells. This combination of methods is used for clinical genetic studies on heterozygote detection in Fabry's disease, an X‐linked genetic disease. More‐over, microchemistry is used to study enzyme activities in sorted autofluorescent “aged” fibroblasts.
Anion transport heterogeneity detected by flow cytometric measurement of NBD‐taurine efflux kinetics
Authors: Muirhead K. A. , Steinfeld R. C. , Severski M. C. , Knauf P. A. 
Published: August 2005

AbstractNBD‐taurine [N‐(7‐nitrobenzofuran‐4‐yl) taurine], a fluorescent substrate for the human erythrocyte anion exchange system, has been used to test the feasibility of making flow cytometric measurements of anion transport in K562 erythroleukemic cells. Cells were preloaded by incubation with 20 μM‐2mM NBD‐taurine, then diluted 10–30‐fold, and efflux was monitored by measuring fluorescence intensity (FL) as a function of time using excitation at 488 nm. The observed rate of decrease in fluorescence was sensitive to temperature and also to phloretin, a compound known to inhibit anion transport and other carrier‐mediated transport processes. The coefficient of variation (CV) of the fluorescence distribution increased markedly over the efflux period, suggesting heterogeneity of the K562 population with respect to the rate constant for NBD‐taurine efflux. This heterogeneity was also reflected in the upward curvature of a first order plot of log (FLt–FL∞) versus time. Half‐times calculated from initial linear portions of the first‐order plots were found to decrease as the loading concentration of NBD‐taurine was decreased, as predicted for a saturable transport system.NBD‐taurine is not an ideal anion transport substrate for flow cytometric studies. It appears to bind to high‐affinity sites within the cells with consequent fluorescence quenching, complicating interpretation of kinetic curves at low concentrations. However, with the appropriate choice of substrate, flow cytometric estimates of kinetic parameters offer a number of advantages over bulk methods: (1) decreased sample size requirements, particularly at low substrate concentrations, (2) sensitive detection of transport heterogeneity as time‐dependent changes in CV and (3) the possibility of sorting subpopulations with different transport parameters for correlation with state of differentiation or cellular physiology.
Reduced insulin endocytosis in serum‐transformed fibroblasts demonstrated by flow cytometry
Authors: Murphy Robert F. , Powers Scott , Cantor Charles R. , Pollack Robert 
Published: August 2005

AbstractNeoplastic transformation often results in the loss of growth control and concomitant changes in cell surface properties. The changes in endocytosis of a variety of probes after serum or anchorage transformation were measured for mouse fibroblasts by flow cytofluorometry. No major differences in dextran (fluid phase) or histone (nonspecific‐adsorptive) endocytosis were observed among four cell lines with different growth properties. However, decreased receptor‐mediated internalization of α‐macroglobulin was observed for cell lines transformed to either serum or anchorage independence. Furthermore, increased wheat germ agglutinin and decreased insulin endocytosis were observed, but only in serum transformants. The changes specific to serum transformants were not accounted for by changes in binding of wheat germ agglutinin or insulin. The possible implications of these observations regarding serum transformation and the insulin requirement for growth in serum‐free medium are discussed.
Incorporation of 35S‐sulfate and 3H‐glucosamine into heparan and chondroitin sulfates during the cell cycle of B16‐F10 cells
Authors: Blair Owen C. , Sartorelli Alan C. 
Published: August 2005

AbstractChanges in glycosaminoglycan composition occurring during the cell cycle were determined in B16‐F10 cells sorted flow cytometrically with respect to DNA content. Incorporation of 35S‐sulfate into heparan sulfate and chondroitin sulfate of unsorted and G1, S, and G2 + M sorted cells was determined following chondroitinase ABC or nitrous acid treatment; the incorporation into surface material was measured as the difference between the radioactivity of control and trypsin‐treated cells. Incorporation of 35S‐sulfate and 3H‐glucosamine into cetyl pyridinium chloride (CPC)‐precipitable material was characterized before and after chondroitinase or nitrous acid treatment by Sephadex G50 chromatography. Long‐term (48 h) and short‐term (1 h) labeling studies demonstrate that (a) the amount of total cellular chondroitin sulfate is greater than that of heparan sulfate, with larger amounts of unsulfated heparan than chondroitin being present; (b) the rate of turnover of heparan sulfate is greater than that of chondroitin sulfate; (c) greatest short‐term incorporation of 3H‐glucosamine into CPC‐precipitable material occurs during S phase; and (d) the rate of turnover of both heparan sulfate and chondroitin sulfate is decreased in S phase relative to G1 and G2+ M.
An ultra‐pure in vitro phase synchrony method employing centrifugal elutriation and viable flow cytometric cell sorting
Authors: Rice G. C. , Dean P. N. , Gray J. W. , Dewey W. C. 
Published: August 2005

AbstractWe present a method of synchronizing cells in G1‐, S‐, and G2M‐phases employing sequential centrifugal elutriation and viable flow cytometric cell sorting of Hoechst‐33342 stained Chinese hamster ovary cells. G1‐ and S‐phase cells can be separated to greater than 99% homogeneity and G2‐M to 70% purity. Most of the 30% contamination in the G2‐M fraction was due to S‐phase cells, whose reproductive integrity could be eliminated through the use of high specific activity 3H‐TdR. There were minimal toxic effects or perturbations to growth following the selection procedures. The most significant limitation of this technique appears to be the rate of cell sorting, which, with current equipment, is approximately 3,000 cells per second.
The use of projections for dimensionality reduction of flow cytometric data
Authors: Mann R. C. , Popp D. M. , Hand R. E. 
Published: August 2005

AbstractIn order to visualize multiparameter flow cytometric measurements it is desirable to reduce the dimensionality of the data to two while preserving important features of each data pattern. We describe a method that projects two‐parameter data onto a straight line that forms an angle θ with one of the original parameter axis. The angle is selected interactively or by principal component analysis. The procedure is applied on‐line or off‐line to pairs of parameters of the original data set that has been collected in LIST mode.
New fluorochromes, compatible with high wavelength excitation, for flow cytometric analysis of cellular nucleic acids
Authors: Latt Samuel A. , Marino Michael , Lalande Marc 
Published: August 2005

AbstractThe spectroscopic properties of three new dyes, EK4, VL772, and LL585, free and bound to nucleic acids, are presented, with particular emphasis on their potential use in flow cytometry. Two of these dyes, EK4 and LL585, exhibit red fluorescence, while dye VL772 exhibits yellow fluorescence. Dye LL585 exhibits specificity for DNA, relative to RNA, and a marked enhancement of fluorescence efficiency upon binding to DNA, needed for a red fluorescent DNA‐specific stain for flow cytometry. The dye penetrates live cells, although uniformity of nuclear fluorescence, as evidenced by DNA flow histograms, is better if the cells are first permeabilized with Triton X‐100. Dye VL772 exhibits yellow fluorescence and little DNA‐RNA discrimination, but may prove useful in conjunction with dye LL585 when simultaneous assay of cellular RNA and DNA is desired. Dye EK4 shares properties of the other two dyes but fluoresces with much less efficiency. Dyes LL585 and VL772, used singly, as a pair, or in combination with blue‐fluorescing DNA specific dyes, such as bisbenzimidazole derivatives, should permit new, convenient analyses of the content and organization of cellular nucleic acids.
The relative effects of different types of growth factors on DNA replication, mitosis, and cellular enlargement
Authors: Zetterberg Anders , Engström Wilhelm , Dafgård Eva 
Published: August 2005

AbstractIt was recently demonstrated that growth in cell size can be dissociated from DNA synthesis and mitosis. 3T3 cells starved to quiescence in low serum concentration can be stimulated to undergo DNA synthesis and one cell division without growing in size (unbalanced growth) (42–44). We report here that in cells stimulated to undergo unbalanced growth, the cell nucleus undergoes balanced growth, i. e., nearly doubles in size prior to mitosis. The reduced ability to grow in cell size under unbalanced growth conditions is thus mainly ascribable to the cytoplasm. Furthermore, the extent to which cells grow in size prior to mitosis is dependent on the serum concentration in the tissue culture medium (44).This data suggests that some macromolecular factor or factors in serum are required for growth in cell size prior to mitosis. We report in this study that epidermal growth factor (EGF) alone exerts a small but significant stimulatory influence on DNA synthesis and mitosis but does not affect cellular enlargement. In contrast, insulin added at supraphysiological concentrations does not stimulate quiescent cells to enter S phase but instead stimulates growth in cell size in the small fraction of dividing cells. Furthermore, cells stimulated to proliferate by EGF could be induced to undergo balanced growth when insulin was added concomitantly. Finally, platelet‐derived growth factor (PDGF) stimulates quiescent sparse 3T3 cells to undergo DNA synthesis and mitosis. PDGF also exerts a limited but significant effect on cellular enlargement. However, PDGF alone could not induce a complete balanced growth, i. e., a doubling in cell size prior to mitosis.
The DNA‐based human karyotype
Authors: Mayall Brian H. , Carrano Anthony V. , Moore Dan H. , Ashworth Linda K. , Bennett Deborah E. , Mendelsohn Mortimer L. 
Published: August 2005

AbstractImage cytometry and computer analysis are used to determine the relative DNA content and the DNA‐based centromeric index of the 24 chromosomes of the human karyotype. A two‐step procedure is used. Chromosomes of cells in metaphase first are stained with quinacrine and identified visually by their fluorescent Q‐band patterns. They then are stained for DNA using gallocyanin‐chrome alum. The chromosome images are scanned and recorded as digital values of optical density by an CYDAC image cytometric microscope system, CYDAC. The digital images are processed by computer to measure for each chromosome the relative DNA stain contents of the whole chromosome and of the p and q arms and the DNA‐based centromeric index. About ten cells are analyzed for each of the donors, who are phenotypically normal men and women. The chromosome measurements are pooled by chromosome type for each donor and are compared among donors. The means of the chromosome measurements give the DNA‐based human karyotype. Analysis of the DNA‐based data shows that some chromosomes or portions of chromosomes vary significantly among donors. These variants do not correlate with detectable morphologic polymorphisms, such as Q‐ or C‐band variants; thus they represent new and otherwise undetectable chromosome polymorphisms whose genetic basis and clinical significance are yet to be determined.
The relationship between thrombomodulin expression and cell cycle stages in cultured rabbit endothelial cells
Authors: Debault Lawrence E. , Esmon Naomi L. , Esmon Charles T. 
Published: August 2005

AbstractCultured rabbit endothelial cells have significant but variable amounts of thrombomodulin (TM), both on their surface as well as inside the cell. To determine if variations in TM antigen is cell cycle related, cells with very high levels of TINI antigen were identified and staged according to the intracellular distribution and relative amounts of the antigen, using immunofluorescence techniques. After staging, the nuclear DNA content of each of these cells was determined by measuring the propidium iodide (PI) fluorescence intensity cytophotometrically. Stages 1, 2, and 3, which exhibited TM immunofluorescence in the golgi area, clustered to the G1 phase of the cell cycle. Cells without discernible golgi fluorescence (stages 4 and 5) but with variable amounts of cytoplasmic and surface fluorescence appeared to have little or no relationship to the cell cycle.
Quantitative histochemistry of the angiotensinase A (APA) in the renal glomeruli of rats after stimulation of the renin‐angiotensin system,
Authors: Kugler P. , Schiebler T. H. 
Published: August 2005

AbstractQuantitative histochemical measurements of aminopeptidase A(APA, angiotensinase A) were clone kinetically in the kidney glomeruli of rats after short‐term experiments (treatment with furosemide as well as captopril for 2, 4, and 6h). The APA activities increased after treatment with furosemide or captopril. Highest activities were determined after 4h using furosemide and 6h using captopril. It is concluded that glomerular APA activities correspond to the renin/angiotensin plasma levels and that the fast changes of APA activities are well demonstrable by kinetic densitometric measurements in situ.
The effects of naturally occurring metabolites (L‐cysteine, vitamin C) on cultured human cells exposed to smoke of tobacco or marijuana cigarettes,
Authors: Leuchtenberger Cecile , Leuchtenberger Rudolf 
Published: August 2005

AbstractThe effects of vitamin C on the growth of human lung cultures and of vitamin C or L‐cysteine on a human breast cancer culture (SK‐Br‐3) were assessed with and without exposure to fresh smoke from tobacco or marijuana cigarettes. When grown in the presence of vitamin C, lung cultures exposed or not exposed to either type of smoke showed a stimulation of growth and a significant decrease in mitotic abnormalities. However, abnormalities were much more marked in marijuana‐exposed cultures than in tobacco‐smoke‐exposed ones. Nonexposed or tobacco‐smoke‐exposed breast cancer cultures, when grown in the presence of vitamin C, also showed acceleration of growth of epithelial cells, significant reduction in mitotic abnormalities, and occurrence of pseudoglandular structures, indicating differentiation. These alterations not only disappeared, but the cultures also became fibroblastic when they were returned to media without vitamin C. In contrast, vitamin C did not reduce mitotic abnormalities in marijuana‐smoke‐exposed breast cancer cultures, but stimulated abnormal growth and dedifferentiation. In nonexposed, tobacco, or marijuana‐smoke‐exposed breast cancer cultures, L‐cysteine evoked an acceleration of fibroblastic growth, which was not altered when the cultures were returned to media without L‐cysteine.