bluuebunny/crossref_metadata_2025_split huggingface dataset:
Flow cytometric analyses of lectin binding to rat alveolar macrophages
Authors: Saunders George C. , Steinkamp John A. , Lehnert Bruce E. 
Published: August 2005

AbstractThe binding characteristics of ten FITC‐labeled plant lectins (Con‐A, MPA, BPA, PNA, WGA, SBA, UEA‐I, DBA, GS‐I, GS‐II) to lavaged rat alveolar macrophages were assessed by flow cytometry. The alveolar macrophages (AM) were incubated with varying concentrations of each lectin in a pinocytosis‐inhibiting buffer. In addition to measuring lectin‐associated green fluorescence, the electronic cell volumes and axial light loss characteristics of the AM were also measured flow cytometrically. These latter parameters were found to be good indicators of cell agglutination caused by some of the lectins, and, in conjunction with green fluorescence measurements, usefully serve to determine optimal or nonagglutinating lectin concentrations for flow cytometric studies. With the exception of UEA‐I, all of the lectins examined bound to AM, although a wide range of binding was observed among the lectins. At subagglutinating concentrations, Con A, MPA, BPA, PNA, WGA, SBA, and GS‐I bound to the AM with unimodal patterns. Histograms of lectin‐associated fluorescence intensity obtained with DBA clearly presented a pattern consistent with a more complex, bimodal distribution of labeled AM, suggesting the presence of at least two subset populations. The low‐intensity distribution of AM represented ≃ 70% of the cells, while the more strongly labeled subset represented ≃ 15% of the parent AM population. The remaining balance of the AM was identified as another subpopulation by the failure to detectably bind to the DBA. While GS‐II bound to all of the AM, this lectin labeled about 5% of the cells much more intensely than the bulk of the population. Thus, two subset populations of AM could be resolved according to their differing avidities for the GS‐II lectin.
Improved program for the analysis of DNA histograms
Authors: Ormerod M. G. , Payne A. W. R. , Watson J. V. 
Published: August 2005

AbstractWe describe a computer program which will rapidly analyze a set of DNA histograms without further operator intervention after the initial input. The algorithm used was modified from that of Watson et al. (Cytometry 8:1–8, 1987). The program, coded in Pascal, has been implemented on two different systems. It has been tested by using simulated data and experimental data from cells labelled with bromodeoxyuridine.The program has been in use in this and other laboratories for over two years and has proved to be robust and fast and to give a reasonable approximation to the experimental data. It is particularly useful when dealing with badly perturbed DNA histograms.
Four‐Parameter white blood cell differential counting based on light scattering measurements
Authors: Terstappen L. W. M. M. , DeGrooth B. G. , Visscher K. , VanKouterik F. A. , Greve J. 
Published: August 2005

AbstractMeasurement of the depolarized orthogonal light scattering in flow cytometry enables one to discriminate human eosinephilic granulocytes from neutrophilic granulocytes. We use this method to perform a four‐parameter differential white blood cell analysis.A simple flow cytometer was built equipped with a 5‐mW helium neon laser that measures simultaneously four light scattering parameters. Lymphocytes, monocytes, and granulocytes were identified by simultaneously measuring the light scattering intensity at angles between 1.0° and 2.6° and angles between 3.0° and 11.0°. Eosinophilic granulocytes were distinguished from neutrophilic granulocytes by simultaneous measurement of the orthogonal and depolarized orthogonal light scattering.Comparison of a white blood cell differentiation of 45 donors obtained by the Technicon H‐6000 and our instrument revealed good correlations. The correlation coefficients (r2) found were: 0.99 for lymphocytes, 0.76 for monocytes, 0.99 for neutrophilic granulocytes, and 0.98 for eosinophilic granulocytes. The results demonstrate that reliable white blood cell differentiation of the four most clinically relevant leukocytes can be obtained by measurement of light scattering properties of unstained leukocytes.
Simultaneous cytometric analysis for the expression of cytoplasmic and surface antigens in activated T cells
Authors: Hayden Georgina E. , Walker Karen Z. , Wotherspoon John S. , Raison Robert L. , Miller Jacques F. A. P. 
Published: August 2005

AbstractA method of two‐colour immunofluorescence staining has been developed to allow the simultaneous analysis of both surface and cytoplasmic antigens. This involves the use of direct fluorochrome antibody conjugates for cell‐surface antigen staining, followed by cell permeabilization and the staining of cytoplasmic antigens with biotinylated antibodies and streptavidin‐fluorochrome conjugates.Fluorochrome‐antibody conjugates bound to cell‐surface epitopes were found not to be affected by the subsequent permeabilisation and cytoplasmic staining. This method was used to examine the surface phenotype of T cells expressing a cytoplasmic antigen, STA. STA is a unique determinant detected in activated human T cells by the monoclonal antibody K‐1‐21, which also recognizes a cross‐reactive conformation‐dependent epitope on human free kappa light chains. Cytometric analysis showed that STA is found in both Leu 2a+ cytotoxic/suppressor T cells and Leu 3a+ helper/inducer T cells but is not induced in the Leu 15+ population which contains suppressor T cells. STA was also shown to be an activation antigen in murine T cells.
Analysis of simian virus 40 infection of CV‐1 cells by quantitative two‐color fluorescence with flow cytometry
Authors: Lehman John M. , Laffin Judith , Jacobberger James W. , Fogleman David 
Published: August 2005

AbstractQuantitative two‐color fluorescent analysis of Simian virus (SV40) infection of permissive CV‐1 cells was investigated. Analysis included by quantitation of cellular DNA, the early viral tumor (T) antigen with a monoclonal antibody, and late viral (V) antigens with a polyclonal antibody. T antigen was detected in all phases of the cell cycle at 6 and 12 h, after SV40 infection of growth arrested cells. At later time intervals, the percentage of T‐antigen‐positive cells increased with the induction of the cells into successive rounds of DNA synthesis and an increase in tetraploidpolyploid cells. The amount of T antigen per cell increased as the cells entered the successive stages of the cell cycle (G0/G1 → G2 + M → tetraploid S and G2 + M). The V antigen from adsorbed virus was detected immediately after infection. Synthesis of V antigen began in late S and G2 + M phases of the cell cycle. This quantitative analysis allows a definitive determination of antigen per cell in a population correlated with the cell cycle and may be useful in correlating viral and cellular events with transformation.
Transmission electron microscopy of small numbers of sorted cells
Authors: Sebring R. J. , Johnson N. F. , Spall W. D. 
Published: August 2005

AbstractA new method that allows the transmission electron microscopic examination of as few as 1 × 104 cells obtained by flow cytometric sorting is described. The approach involves “sandwiching” fixed cells in an agarose case by a microcentrifugation system consisting of small‐diameter cell‐centrifugation tubes and subsequent processing of the cells by conventional techniques. The advantages offered by this method are discussed.
Acousto‐optic laser‐scanning cytometer
Authors: Burger Douglas , Gershman Russell 
Published: August 2005

AbstractAn instrument has been developed that uses a computer‐controlled rapidly scanning laser beam to make cytometric measurements on cells or particles and which can measure low levels of fluorescence when using low‐power lasers (Gershman, Hoffman, and O'Connell, “Methods and Apparatus for Analysis of Particles and Cells.”) The method used is based upon acousto‐optic principles of light diffraction. A vertically polarized 5‐mW He‐Ne laser is directed into an acousto‐optic Bragg cell in which a portion of the incident light under goes a small angular variation or deflection. Suitable optics focus the beam to a 25 μm diameter spot, at the 1/e2 point, in a sample cuvette while translating the angular variation into a linear scan. The cuvette enclosing the sample is slowly moved (approximately 1 μm/ms) via a stepper drive into the scanning beam while the forward angle light scatter sensor is monitored for the presence of valid signal events. When an event occurs, appropriate software optimizes the position of the focused laser beam onto the cell. Subsequently, scanning is stopped to allow for cell interrogation times that last for milliseconds or longer.
Discrimination of myogenic and nonmyogenic cells from embryonic skeletal muscle by 90° light scattering
Authors: Yablonka‐Reuveni Zipora 
Published: August 2005

AbstractA myogenic cell suspension was isolated from the breast muscles of 10‐day‐old chicken embryos by trypsin digestion. The cell preparation was subjected to Percoll density centrifugation to reduce the number of fibroblastlike cells present. The Percoll‐isolated, predominantly myogenic cell population was then fractionated by flow cytometry using 90° light scattering as the parameter for sorting. Cells exhibiting lower scatter, with a peak of 45 units, produced cultures containing myotubes and gave rise only to myogenic clones. Cells exhibiting higher scatter (120‐200 units) produced nonmyogenic cultures and gave rise to nonmyogenic clones. Cells with intermediate light scatter were also detected. The latter produced both myogenic and nonmyogenic clones. The differences in light scatter presumably reflect higher cytoplasmic complexity of the nonmyogenic cells compared with the myogenic cells. Moreover, the differences in light scattering properties of the different cell types offer a means for the isolation of pure populations of myogenic cells directly from the intact muscle.
Flow cytometric measurements of cytoplasmic calcium changes in human platelets
Authors: Davies Theresa A. , Drotts Daniel , Weil Gary J. , Simons Elizabeth R. 
Published: August 2005

AbstractThrombin‐induced stimulation of human platelets is accompanied by a dramatic increase in cytoplasmic calcium concentrations followed by a slow decrease. These changes are very rapid, are maximal by 10‐5 s, and can be detected with probes such as Indo‐1. Suspension studies using spectrofluorometry, which reflect a value which is the average of 3 x 107 cells per ml, indicate a thrombin dosedependent increase in cytoplasmic calcium at doses up to 0.025 units per ml. We show here, using flow cytometry, that at less than halfsaturating thrombin doses only subpopulations of platelets rather than the entire sample are responding. The extent of these responses, however, still depends on thrombin concentration. When the thrombin doses are between half and fully saturating, one subpopulation responds fully (i.e., its extent of increase in cytoplasmic calcium concentration, [Ca++]in is 100% of that seen at saturating thrombin concentrations) while the remaining platelets respond partially or not at all. There is thus evidence of positive cooperativity leading to disproportionate thrombin receptor occupancy on different subpopulations when platelets are subjected to subsaturating doses of thrombin. The existence of responding subpopulations may explain how the reported multiple stimulations of the same suspension of platelets at low thrombin doses occur.
Comparison of fresh and paraffin‐embedded tissue as starting material for DNA flow cytometry and evaluation of intratumor heterogeneity
Authors: Kallioniemi Olli‐Pekka 
Published: August 2005

AbstractFlow cytometric analysis of DNA ploidy and S‐phase fraction (SPF) from paraffin‐embedded tumors has become an important diagnostic and prognostic tool in clinical pathology and investigative oncology. The present study aimed at elucidating the reliability of the method. About 90% of the 1,400 paraffine‐mbedded tumors analyzed were evaluable for DNA index and about 70% for SPF, although considerable differences between various tumor types were detected. The within‐assay coefficients of variation for determination of tumor DNA index and SPF were 2% and 6%, respectively. Intratumor variation in DNA index was observed in 24% of breast and in 21% of ovarian carcinomas and variation in SPF in 36% and 29% of the respective tumors. Intratumor variation in SPF was greatest in DNA‐diploid tumors, which may indicate that SPF values in these tumors may be less reliable owing to variations in the proportions of tumor and nontumor cells. If the methodological variation and the intratumor variation were taken into account, there was a good correlation between DNA indices (r = 0.980) and between SPF values (r = 0.794) obtained from fresh and paraffin‐embedded tumors. It is concluded that accurate determination of DNA index and SPF from paraffin‐embedded tumors is possible in the majority of cases. Regardless of the type of starting material used for DNA flow cytometry it is advantageous to study several samples from each tumor to account for the intratumor heterogeneity.
Flow cytometric discrimination of mitotic nuclei by right‐angle light scatter
Authors: Zucker Robert M. , Elstein Kenneth H. , Easterling Robert E. , Massaro Edward J. 
Published: August 2005

AbstractFlow cytometry has been used to demonstrate alterations in protein, RNA, and DNA content of cells as they traverse the cell cycle. Employing fluorescein isothiocyanate (FITC) to stain protein and propidium iodide (PI) to stain nucleic acids, multiple regions within the G1 and G2 phases of the cell cycle, in addition to the M phase, can be distinguished. In this study, cytograms of the 90° light scatter signal vs. PI fluorescence were remarkably similar to those of FITC fluorescence vs. PI fluorescence, suggesting a relationship between 90° light scatter and protein content. M‐phase nuclei can be distinguished from G2‐phase nuclei on cytograms of 90° light scatter vs. PI fluorescence. However, the percentage of mitotic nuclei obtained by this technique is less than that found by light microscopic analysis. Flow cytometric parameters of nuclei prepared by nonionic detergent (NP40) lysis in Dulbecco's PBS, Vindelov's buffer, or Pollack's hypotonic EDTA/Tris buffer were compared. The best resolution of mitotic nuclei was obtained in Pollack's buffer. However, the stainability of the M‐phase nuclei is reduced, and the nuclei are located in the late S/G2 region of the single‐parameter histogram.
Detection of an early cytomegalovirus antigen with two‐color quantitative flow cytometry
Authors: McSharry James , Laffin Judith , Fogleman David , Lehman John M. , Elmendorf Sarah 
Published: August 2005

AbstractAn early cytomegalovirus (CMV) antigen was detected with a monoclonal antibody by two‐color fluorescent flow cytometry. With the aid of a human diploid fibroblast cell strain, FLOW 2000, infected with the AD169 strain of CMV, the viral antigen and the DNA content of infected or uninfected cells were measured. There was no evidence of change in the cell‐cycle distribution of the infected cells. The viral antigen was detected within 30 minutes following virus adsorption at 0.1 and 1.0 plaque‐forming units/cells; and the percentage of positive cells increased with time and viral dosage. All stages of the cell cycle were susceptible to viral infection and the average fluorescence was greater than the background fluorescence. Flow cytometry detected the viral antigen earlier than conventional immunofluorescent microscopy and cell culture for CMV cytopathological effect (CPE). Ten bronchoalveolar lavages assayed by flow cytometry and conventional diagnostic procedures demonstrated that flow cytometry might be useful in early diagnosis for CMV infection.
A correction required for calculation of DNA ratios in flow cytometric analysis of ploidy
Authors: Givan Alice L. , Shenton B. K. , Carr T. W. 
Published: August 2005

AbstractFlow cytometric measurements of nuclear DNA content often involve the calculation of a DNA index, which compares the DNA fluorescence from two different populations. Such DNA indices have been used to classify aneuploid peaks from tumour tissue into different categories relative to normal diploid cells. This report describes a correction based on the channel for unstained particles that is required if DNA index values are to give a true and reproducible indication of relative DNA content.
Comparison of frequency distributions in flow cytometry
Authors: Cox Christopher , Reeder Jay E. , Robinson Roy D. , Suppes Sarah B. , Wheeless Leon L. 
Published: August 2005

AbstractA number of methods have previously been considered for the statistical comparison of flow cytometric frequency distributions. For two distributions, the foremost of these is the Kolmogorov‐Smirnov (K‐S) test, which has been criticized as “too sensitive.”We discuss some alternative methods based on the Poisson distribution. The assumption of Poisson variation within channels allows the use of channel‐by‐channel confidence intervals and chi‐square tests. These are simple and more appropriate for discrete data than the K‐S test. Graphical displays of these and other techniques are presented. We also attempt to set the problem in an appropriate context. We argue that any statistical procedure must rest on a reasonable understanding of the nature of the variability in the system. This understanding takes the form of an appropriate probability model, which may be approximate but must provide a reasonably accurate description of the data. Incomplete understanding of the data can lead to inappropriate analysis. We discuss the assumptions that underlie our techniques and consider extensions to more complex situations.
Identification of cell subpopulations by dual‐color surface immunofluorescence using biotinylated and unlabeled monoclonal antibodies
Authors: Cohen J. H. M. , Aubry J. P. , Banchereau J. , Revillard J. P. 
Published: August 2005

AbstractA new method of dual‐color immunofluorescence is presented for analysis of surface antigen distribution among heterogeneous cell suspensions. It involves flow cytometric analysis of cells stained with a biotinylated first monoclonal antibody and/or with an unlabeled second monoclonal antibody. After addition of streptavidin‐phycoerythrin and/or fluoresceinated goat antimouse immunoglobulin antibody, single‐cell fluorescence intensties are measured and biparametric graphic representations are obtained, allowing one to determine the percentage of cells stained by each of the monoclonal antibodies or both. The validity of the method was assessed on human peripheral blood mononuclear cells by using three sets of two monoclonal antibodies: CD8 and CD5, CD3 and CD4, CD11 and HLA‐DR. The results showed that dual staining did not induce significant quenching or competition between pairs of antibodies. The procedure is simple and sensitive. It requires only minute amounts of monoclonal antibodies. It is readily applicable to the screening of hybridoma supernatants and to the characterization of new antibodies to cell surface antigens with respect to well‐defined markers.
Chromatin texture changes related to proliferation and maturation in erythrocytes
Authors: Giroud Françloise , Gauvain Catherine , Seigneurin Daneil , Hagen Victoria Von 
Published: August 2005

AbstractErythroid cell types were quantitatively assessed on the basis of the analysis of their nuclear images after Feulgen and Heidenhain blue counterstaining. An original nonsupervised classification method, developed in our laboratory, was applied to cell cycle analysis. Results obtained suggest that, in the four proliferative generation cycles, which concur with the successive morphological stages, there is a sequential flow of cells from one morphological stage to the next by cell cycling. We show that the chromatin pattern alone made it possible to set up a metric of the whole differentiation process occurring in the normal human erythroblastic lineage in which proliferating events occur.
Light regulation of the cell cycle in Euglena gracilis bacillaris
Authors: Yee Muh‐Ching , Bartholomew James C. 
Published: August 2005

AbstractWe have studied the light regulation of the cell division cycle in the photosynthetic alga Euglena gracilis bacillaris. Euglena grown under phototrophic conditions are easily synchronized to a 12 h light‐12 h dark regime. By inoculating stationary phase, nondividing cells into fresh media and exposing the diluted cells to either light or darkness, we have determined that initiation of DNA synthesis for the cell division cycle is light dependent. By varying the length of time in light to which synchronized cells are exposed, we have shown that commitment to the cell cycle requires exposure to more than 6 h of light. We propose that this is to allow the accumulation, through photosynthetic electron transport, of an initiating factor that will enable DNA synthesis to begin. Flow cytometry analysis also shows that once cells are committed to the cell cycle, they complete the cycle in the dark, so mitosis is a light‐independent step.
Interval‐coded texture features for artifact rejection in automated cervical cytology
Authors: Tucker James H. , Rodenacker Karsten , Juetting Uta , Nickolls Peter , Watts Keith , Burger Georg 
Published: August 2005

AbstractIn order to improve the separation between abnormal cells and noncellular artifacts in the CERVIFIP automated cervical cytology prescreening system, 22 different object texture features were investigated. The features were all statistical parameters of the pixel density histograms or one‐dimensional filtered values of central and border regions of the object images. The features were calculated for 231 images (100 cells and 131 artifacts) detected as Suspect Cells by the current CERVIFIP and were then tested in hierarchical and linear discriminant classifiers. After selecting the two best features for use in a hierarchical classifier, 83% correct classification was achieved. One of these features was specifically designed to remove poorly focused objects. With maximum likelihood discrimination using all 22 features, an overall correct classification rate of 90% was obtained.
In vitro aging of articular chondrocytes identified by analysis of DNA and tubulin content and relationship to cell size and protein content
Authors: Ronot Xavier , Gaillard‐Froger BÉAtrice , Hainque Bernard , Adolphe Monique 
Published: August 2005

AbstractIn vitro senescence of chondrocytes, characterized by a decline in the proliferation rate during late passages, resulted from a rapid growth rate in early subcultures to a complete loss of division after seven to nine passages. One senescent‐associated phenotypic change was the apparent increase in the density of cytoplasmic cytoskeletal proteins. We examined the relationship between tubulin content and growth (measured by DNA and total protein contents and cell volume), using flow cytometry, in the assessment of cytoskeleton analysis during in vitro aging. In contrast with previous microscopic observations of tubulin organization, flow cytometry revealed a tubulin content that was modulated as a function of protein content and/or cell volume.