bluuebunny/crossref_metadata_2025_split huggingface dataset:
Flow cytometric evaluation of cell cycle characteristics during in vitro differentiation of chick embryo chondrocytes
Authors: Giaretti W. , Bruno S. , DiVinci A. , Geido E. , Moro G. , Quarto R. , Cancedda R. 
Published: August 2005

AbstractThe cell cycle kinetic characteristics of chick endochondral chondrocytes differentiating in vitro were studied by flow cytometry. In addition, the synthesis of type I and type X collagens of the same cells was evaluated by immunoprecipitation. Dedifferentiated cells, derived from chick embryo tibiae and grown attached to a substratum, were characterized by type I collagen synthesis, a high growth fraction (GF = 0.94), minimal cell loss factor (ϕ = 0.02), and a total cell cycle time of the proliferating cells of about 17 h (tG1 = 8 h, tS = 5 h, and tG2 + M = 4 h). Transfer of dedifferentiated cells to suspension culture on agarose‐coated dishes induced differentiation to hypertrophic chondrocytes. These were characterized by type X collagen synthesis, a low growth fraction (GF = 0.52), maximal cell loss factor (ϕ = 1.0), and a total cell cycle time of the proliferating cells of about 73 h (tG1 = 53 h, tS = 12 h, and tG2 + M = 8 h). The transition from dedifferentiated chondrocytes to hypertrophic chondrocytes was accompanied by large increases of the duration of all the cell cycle phases and of the number of quescent and degenerating cells. Associated with these alterations in cell cycle kinetics was a switch from type I to type X collagen synthesis. Further preliminary data suggest that the population of differentiating chondrocytes (a state between dedifferentiated and hypertrophic chondrocytes) comprises a heterogeneous population of fast and slow growing cells.
Flow cytometric analysis of DNA content differences in blood samples obtained by leucoconcentration
Authors: Pierrez J. , Guerci A. , Guerci O. 
Published: August 2005

AbstractThe leucoconcentration technique allows rapid obtainment of cellular suspensions from total blood or bone marrow for flow cytometric analysis. The technique is based on picric acid in ethyl alcohol fixation and saponin red cell lysis, followed by mithramycin staining for DNA. It gives a good resolution of DNA distributions that allow detection of slight variations in DNA content. These results were obtained with cellular suspensions differeing only in one X or Y chromosome (male, female, Klinefelter and Turner syndromes).In these studies the ratio of the DNA content of X and Y chromosomes agrees with the chromosomal mass ratio already reported by other authors, but the “absolute values” are 10‐fold more compared to these same works. Our conclusion is that leucoconcentration technique followed by DNA staining with mithramycin increases the difference in the dye's penetration and binding between X and Y chromosomes.
Two‐Color flow cytometric analysis of monocyte depleted human blood lymphocyte subsets
Authors: Fleisher Thomas A. , Hagengruber Claire , Marti Gerald E. 
Published: August 2005

AbstractPeripheral blood mononuclear cells (PBMC) from normal individuals were examined using 16 pairs of FITC and phycoerythin (PE) directly conjugated monoclonal antibodies. Each pair of reagents was used to evaluate a conventional lymphocyte gate as well as open (non) gate of monocyte depleted PBMC. Parallel studies using the same panel of monoclonal antibodies were carried out on selected, nonmonocyte depleted samples. The major findings of this analysis were that 1,000–1,200 lymphocytes in a 10,000 cell analysis are found outside the lymphocyte gate and of these approximately 2/5 are CD16 positive LGL/NK cells, 2/5 are CD3 positive T cells, and 1/5 are CD19/CD20 positive B cells. Thus, it appears that 10–15% of the lymphoid cells fall outside of the conventional lymphocyte gate, and in certain settings monocyte depletion may be useful to perform more complete evaluation of the total lymphoid cell population obtained after ficoll‐hypaque separation.
Phagocytosis, intracellular pH, and cell volume in the multifunctional analysis of granulocytes by flow cytometry
Authors: Rothe G. , Valet G. 
Published: August 2005

AbstractPhagocytosis of Escherichia coli K12 strain bacteria was used to measure by flow cytometry the functional activities of human granulocytes in whole blood or buffy coat preparations. In a first measurement, the increase in electric cell volume and acridine orange (AO) green and red fluorescence were used to quantify the degree of phagocytosis. In a second measurement, the intracellular pH and esterase activity of each cell were determined with 1,4‐diacetoxy‐2,3‐dicyanobenzene to obtain information on the metabolic activities during phagocytosis and degradation of bacteria. The DNA of dead cells was simultaneously counterstained with propidium iodide in both assays. The volume, the AO green and red fluorescence, the internal pH, and esterase activity were automatically averaged for all granulocytes or lymphocytes of a measurement. The calculated mean values were transferred into the self‐learning database of the DIAGNOS1‐program system. The functional granulocyte parameters of normal healthy individuals can be used as reference values for the automated diagnosis of abnormal granulocytes in various infectious disease states. The assays require 1 ml of heparinized whole blood and the results are available within 1 hour.
Subpopulation analysis of drug‐induced cell‐cycle delay in human tumor cells using 90° light scatter
Authors: Epstein R. J. , Watson J. V. , Smith P. J. 
Published: August 2005

AbstractA mitotic cell subset has been identified with nuclear light scatter. Colcemid‐treated T‐47D human breast cancer cells were permeabilised, stained with ethidium bromide, and analysed by flow cytometry. Cells with G2M DNA content exhibited a unimodal distribution for DNA fluorescence and forward scatter, but two peaks were discernible with 90° light scatter. A discrete low‐scattering cell cluster could be distinguished from the G2 cell subset on two‐dimensional contour plots of 90° light scatter vs. DNA fluorescence; this cluster was reproduced by mitotic shake‐off experiments and varied quantitatively with mitotic indices determined either by microscopy or by stathmokinetic cell‐cycle analysis of DNA fluorescence. Cell sorting confirmed that the low‐scattering cell cluster comprised predominantly metaphase and anaphase cells.Identification of mitotic cells with this onestep technique enables rapid analysis of druginduced cell‐cycle delay in cell populations with different rates of cell‐cycle traverse. Hence, vincristine‐induced cytostasis is shown to arise in part because of premitotic G2 arrest, whereas etoposide is shown to affect cycling cells with equal sensitivity in quiescent and activated cell populations. The use of light scatter to discriminate mitotic cells in this way facilitates analysis of drug‐induced cell‐cycle delay and supplements the information obtainable by conventional cell‐cycle analysis.
Isolation of highly multidrug‐resistant P388 cells from drug‐sensitive P388/S cells by flow cytometric cell sorting
Authors: Ross Douglas D. , Ordóñez José V. , Joneckis Christopher C. , Testa Joseph R. , Thompson Bruce W. 
Published: August 2005

AbstractTo investigate the spontaneous frequency of occurrence of stable multidrug‐resistant cells in a population of drug‐sensitive cells, we exposed drug sensitive P388/S cells to daunorubicin (dnr) for 1 h, then used fluorescence‐activated cell sorting based on intracellular dnr fluorescence to isolate cells within P388/S having different intracellular content of drug. One of the sort windows chosen (low dnr content sort window) isolated only P388/S cells with intracellular drug content equal to or less than that of the known multidrugresistant subline P388/adr. This sort window constituted approximately 3% of P388/S cells with lowest dnr content. By such a procedure we were able, on one of seven attempts, to isolate and cultivate stable, highly multidrugresistant cells (comparable to that of P388/adr) from the P388/S cells obtained from the low dnr‐content sort window. Net growth of cells in culture was observed 15–20 days after sorting, indicating that of the P388/S cells collected from the low dnr‐content sort window, very few were actually highly drug‐resistant. On no occasion could resistant cells be cultivated from cells sorted from P388/S with higher dnr content, as would be expected if mutation to a multidrug‐resistant phenotype had occurred as a result of exposure to drug. The resistant cells isolated from P388/S by sorting (called P388/LoSort) displayed low intracellular accumulation of dnr that was enhanced by verapamil, were cross‐resistant to vincristine and actinomycin‐D, and distinct from P388/S, possessed a 150‐ to 160–kD membrane species identified by Vinca alkaloid photoaffinity labeling. The cytogenetic alterations found in P388/LoSort were different from those found in P388/adr, confirming that P388/LoSort cells are unique, although phenotypically similar to P388/adr. These studies suggest that spontaneous mutation to a highly multidrug‐resistant phenotype does exist in P388/S cells, but that the mutation frequency is low, with an estimated frequency of 2 × 10−8.
Flow cytometric analysis of the phenotypic changes in tumour cell lines following TPA induction
Authors: Zanyk Marien J. , Banerjee Diponkar , McFarlane David L. 
Published: August 2005

AbstractSingle‐cell analysis by flow cytometry has enabled us to analyze the effects of a phorbol ester and known tumour promoter, TPA, on the phenotypes of four tumour lines. TPA is capable of triggering a variety of cellular alterations that can affect gene expression and the biochemical balance of intracellular events. We have investigated the effect of TPA on such properties as rate of proliferation, differentiation, expression of cell surface molecules, and susceptibility to natural killer (NK) cell‐mediated cytolysis.Four human leukemia and lymphoma cell lines; K562, MOLT 4, Raji, and HL60, were studied in their response to TPA treatment. Based on measurements of the defined cellular properties, we have characterized the pleiotropic responses of each tumour cell line to the phorbol ester in relation to intensity and time of onset of each response. The effects of TPA are highly varied, ranging in time of onset from minutes to days, and in intensity from strong to weak within the four cell lines studied. However, within all the processes that are affected, the activation of protein kinase C appears to be a common initiating event of phorbol ester induction.
A single‐cell assay of β‐galactosidase activity in Saccharomyces cerevisiae
Authors: Wittrup Karl Dane , Bailey James E. 
Published: August 2005

AbstractA novel assay of single‐cell exogenous β‐galactosidase activity in Saccharomyces cerevisiae has been developed. Intracellular fluorescence due to the hydrolysis of resorufin‐β‐D‐galactopyranoside attains a steady state between production of resorufin and its subsequent leakage from the cell. The cells are permeabilized with Triton X‐100, and the assay is performed at 0°C. These conditions were chosen to minimize intercellular fluorescence communication. Free resorufin in the extracellular space is bound by bovine serum albumin to prevent its uptake by cells. Two regimes of fluorescence accumulation are observed, one limited by the rate of diffusion of substrate into the cell, and one limited by the rate of enzymatic cleavage of the substrate. A quantitative correlation between fluorescence and β‐galactosidase activity is obtained under optimized assay conditions.
An interactive multivariate analysis of FCM data
Authors: Kosugi Yukio , Sato Ryuichi , Genka Shigeru , Shitara Nobuyuki , Takakura Kintomo 
Published: August 2005

AbstractThe procedure and results of the interactive multivariate analysis of FCM data are described. Using principal‐components analysis, cluster analysis, and interactive maneuvers, this procedure facilitates an effective data compression from a four‐dimensional space into two‐dimensional space, then allows cluster separation. The procedure is especially effective for separating clusters, which are degenerated in the usual scattergrams. Programs were mostly written in C language on MS‐DOS and were tested on four‐dimensional analysis of the blood cells, which resulted in a successful separation of the degenerated clusters.
Quantitative description of classic and variant small cell lung cancer cell lines by nuclear image cytometry
Authors: Broers J. L. V. , Pahlplatz M. M. M. , Katzko M. W. , Oud P. S. , Ramaekers F. C. S. , Carney D. N. , Vooijs G. P. 
Published: August 2005

AbstractSix small cell lung cancer (SCLC) cell lines were examined using nuclear image analysis to find features characteristic of the classic and the variant type of SCLC. On the basis of their biochemical and biological properties three of these cell lines have been shown to represent the classic types, and three represent the variant type of SCLC. Using a combination of the image‐derived run length, density, and geometric features, it was possible to distinguish between the classic and variant SCLC cell lines.The results of this study may be of help in assessing photometric features for the separation of the classic and variant subtypes of SCLC in solid tumors. Because of differences in treatment and prognosis between these two subtypes, such a separation may be of clinical value.
Differentiation of mitotic melanoma cells from G2 cells and their isolation by use of 5‐bromo‐2′‐deoxyuridine and propidium iodide
Authors: Trinkle Linda S. , Swope Viki B. , Abdel‐Malek Zalfa A. , Nordlund James J. 
Published: August 2005

AbstractThis report describes a method by which mitotic cells were isolated from nonsynchronized Cloudman melanoma cells that had been pulse labeled with 5‐bromo‐2′‐deoxyuridine (BrdUrd) and double‐stained with a fluoresceinated monoclonal antibody to BrdUrd and with propidium iodide (PI). In initial experiments, melanoma cells were first pulse labeled with BrdUrd, treated with prostaglandin E1 (PGE1 10 μg/m1) or vehicle (0.1% ethanol) for up to 24 hours, then stained with anti‐BrdUrd and PI. PGE1‐treated cells monitored at 3‐hour intervals were observed to migrate from S phase to G2 phase, then, enigmatically, back into the late S phase region of the distribution. In other experiments, cells treated with PGE1 were pulse labeled with BrdUrd at the end of the treatment period and harvested. In these experiments, there was a small, discrete subpopulation of cells within the late S phase region of the DNA distribution that was negative for anti‐BrdUrd. This subpopulation of cells was sorted and examined by light microscopy. We observed that 95% of these BrdUrd‐negative “S phase” cells were mitotic cells. Since mitotic cells and G2 cells have equivalent amounts of DNA, the reduced red fluorescence exhibited by these cells may be due to a greater sensitivity to denaturation, which has been described for DNA of mitotic cells, and would account for the phenomenon of cells appearing to move “backwards” in the cell cycle. This report indicates that although the BrdUrd/PI method can further define the cell cycle into four compartments, it can also lead to overestimation of S phase cells in kinetic studies because of contaminating mitotic cells.
Flow cytometric analysis of platelet surface antigens
Authors: Marti Gerald E. , Magruder Louise , Schuette William E. , Gralnick Harvey R. 
Published: August 2005

AbstractA flow cytometric analysis of human platelet surface antigens was carried out using a panel of monoclonal antibody reagents. The reagents used were specific either for the GPIb or the GP IIb/IIIa complex, surface immunoglobulin, or von Willebrand factor (vWf). Indirect surface immunophenotypes were determined using an EPICS V flow cytometer and the monoclonal antibodies 6D1, 10E5, Plt‐1, UR1663, anti‐IgG, and anti‐vWf. Platelets were obtained from normal individuals or patients with either Bernard‐Soulier syndrome (BSS) or Glanzmann's thrombasthenia (GT). Normal platelets were positive for 10E5, 6D1, Plt‐1, and UR1663 and showed negligible activity for anti‐IgG and anti‐vWf. Platelets from individuals with BSS showed a marked reduction in 6D1, while the platelets of a patient with GT showed a marked reduction in binding of 10E5, Plt‐1, and UR1663. Differences between histograms for normal platelets and for platelets from individuals with BSS or GT were evaluated using the Kolmogorov‐Smirnov test. Compared to normal platelets, the BSS and GT platelets contain at least 35‐fold less of the GPIb and GP IIb/IIIa complex respectively. Flow cytometry is a useful and precise method for the study of normal and abnormal surface platelet phenotypes.
Discriminating between damaged and intact cells in fixed flow cytometric samples
Authors: Terstappen L. W. M. M. , Shah V. O. , Conrad M. P. , Recktenwald D. , Loken M. R. 
Published: August 2005

AbstractA vital, nucleic acid stain (LDS‐751) was used to discriminate intact from damaged cells in a flow cytometer even after the samples had been fixed with paraformaldehyde. Three major cell populations with different fluorescence properties with LDS‐751 were found in the fixed samples. Cells not staining or only dimly staining with LDS‐751 were identified as erythrocytes and platelets, respectively. Cells staining with intermediate amounts of LDS‐751 were found to be intact cells, while cells intensively stained were identified as damaged cells. Confirmation of the identity of the populations was obtained by light microscopic examination of the sorted populations and by correlating the fluorescent signals of FDA and LDS‐751 in nonfixed cell preparations. Agglutinated cells could also be identified by the increased fluorescent signal in the LDS‐751 channel as compared with single cells. The spectral properties of this dye permit excitation at 488 nm with emission in the far red portion of the spectrum. This allowed two‐color immunofluorescence to be combined with the intact/damaged cell discrimination on fixed samples. Therefore, intact single cells could be distinguished during flow cytometric analysis, increasing the accuracy of the immunofluorescence measurements. The visualization of the multidimensional data was facilitated using color to discriminate cell populations depicted in multiple perspectives.
Simultaneous analysis of DNA content and surface antigens in human bone marrow
Authors: Hollander Zeev , Loken Michael R. 
Published: August 2005

AbstractIn order to identify when cellular expansion occurs during hematopoietic maturation, a method was developed for the simultaneous analysis of one or two cell‐surface antigens and DNA content on bone marrow cells while preserving their light‐scatter properties. Proliferation in a population defined by light‐scatter and surface‐antigenic characteristics was assessed by measuring the percentage of cells in this population having more than 2C amount of DNA (“proliferation index”). Viable, low‐density (1.077 g/cm3), bone marrow cells, stained with monoclonal antibodies conjugated with fluorescein or phycoerythrin, were fixed with paraformaldehyde and subsequently treated with the detergent, Tween 20. The UV‐excitable DNA stain Hoechst 33342 was used to quantify DNA content in the cells without interference with immunofluorescence. A FACS IV flow cytometer was used, equipped with the first laser at 488 nm emitting for light scattering and immunofluorescence measurements and the second laser emitting at 360 nm for the Hoechst excitation. The Hoechst uptake was the same for all bone marrow populations, yielding a tight coefficient of variation (CV) (average 5.0%) for the G0/G1 DNA peak. This permitted high sensitivity of cell detection in S, G2, and M phases of the cell cycle, while preserving light‐scattering properties of the cells and maintaining cell surface immunofluorescence. The lowest “proliferation index” detected using this technique was 0.08% in a sample obtained from a patient with chronic lymphocytic leukemia. Normal helper T lymphocytes in marrow had approximately 0.5% of the cells in S, G2, or M phase. We show that the erythroid lineage, in the adult normal bone marrow, is the most active in proliferation among all hematopoietic lineages.
An EPROM‐based programmable contour generator for use in flow cytometry
Authors: Wheeless D. M. , Cambier J. L. , Wheeless L. L. 
Published: August 2005

AbstractAn erasable programmable read‐only memory (EPROM) contour generator has been fabricated to produce contours for use in flow cytometry. Contours are analog waveforms representing the fluorescence or light‐scatter intensity distribution along a cell or object. The generator has particular utility in the development and testing of slit‐scan instrumentation and analysis alogrithms. Contours are generated without the requirement of specimens or full operation of the flow instrumentation. The generator provides control of contour height, width, offset, and rate. The EPROM may be custom programmed to produce contours for specific test applications or for reproducing “real” contour events. The generator is useful in situations where constant repetitive contours of predetermined characteristics are required.
Application of bromodeoxyuridine incorporation measurements to the determination of cell distribution within the S phase of the cell cycle
Authors: Khochbin Saadi , Chabanas Agnés , Albert Philippe , Albert Jacques , Lawrence Jean‐Jacques 
Published: August 2005

AbstractFlow cytofluorimetric measurement of incorporated bromodeoxyuridine, using a double‐stained cell population, allows the determination of the distribution of cells along the cell cycle (1). We have developed a simple computer program for the direct treatment of 64 × 64 channel histograms. This analysis appears to provide interesting data about the distribution of cells in the various phases of the cell cycle, namely the S phase.Two examples have been chosen to illustrate possible fields for the application of such a program. Comparison of two cell lines such as friend murine erythroleukemia cells (MELC) and fibroblasts FR3T3 cells has shown that this analysis can be used for cell‐cycle characterization of a given cell line.The program also allows the differential analysis of cell distribution along the cell cycle as a function of a given parameter. This possibility has been applied to study the variation of cell‐cycle parameters as a function of the time of induced differentiation of MELC and reveals changes in the distribution of the cells along the various phases of the cell cycle, namely in the S phase.
Identification of flow‐sorted chromosomes by G‐banding and in situ hybridization
Authors: Rommel B. , Hutter K.‐J. , Bullerdiek J. , Bartnitzke S. , Goerttler K. , Schloot W. 
Published: August 2005

AbstractThe identification of flow‐sorted chromosomes is a very important tool for checking the purity of the fractions obtained. An easy and reproducible method for obtaining G‐banded chromosomes with good resolution of bands is described. Also, we are able to show that the percentage of chromosomes which can be clearly distinguished by this procedure depends to a large extent on the duration of mitotic arrest.In particular when sorting chromosomes from human‐rodent hybrid cell lines, the possibility of using in situ hybridization in addition to conventional staining techniques to characterize the chromosomes can help overcome the problem of highly condensed chromosomes and chromosomal fragments of unknown origin, which cannot be identified otherwise. Thus, we have developed an in situ hybridization technique, based on biotin‐labelled human genomic DNA, which allows a clear distinction between human and rodent chromosomal material to be made.