bluuebunny/crossref_metadata_2025_split huggingface dataset:
Geometrical constraints on the shape of cultured cells
Authors: Heckman C. A. 
Published: August 2005

AbstractPrevious studies on cultured cells revealed that the values of certain shape descriptors were highly correlated despite the fact that there was little mathematical interdependence (Heckman, CA: In Advances in Cell Culture, Vol. 4, K. Maramorosch, ed, pp 85–156, 1985). The independence of such descriptors was tested by calculating the values of correlations among descriptors for a data set consisting of macroscopic objects. The descriptors were unlinked for this data set, confirming that high correlations reflected specific characteristics of cultured cells. These characteristics were identified by constructing model figures incorporating the geometrical features postulated to be responsible for each correlation. Because the position of the ellipse of concentration depended upon the form in which mass was displaced in figures, the fraction of the figure falling within the area of the ellipse (FINE) and the ratio of the ellipse area to the figure's area (ARAT) became decorrelated from each other in figures with massive projections. Further, correlations between ARAT or FINE and variables measuring cavities and inflections of the perimeter showed a complex dependence on the size, shape, and number of invaginations. Descriptors could become decorrelated due to features present in only a minority of cells of a population. Finally, cell populations that differed with respect to physiological characteristics showed no differences in the value of ARAT:FINE correlation. However, correlations between ARAT and the coefficient of variation of centroid‐to‐perimeter distances were altered. This suggested that correlation coefficients may be themselves powerful descriptors of the shape characteristics of cell populations.
Eight‐Parameter PC‐AT based flow cytometric data system
Authors: Kachel Volker , Messerschmidt Roger , Hummel Peter 
Published: August 2005

AbstractAn 8‐parameter flow cytometric data system is described using an IBM‐at compatible personal computer (PC) and a commercial analog to digital conversion (ADC) board. A dedicated pulse processing interface adapts the flow cytometric pulses to the ADC board and controls the number of parameters to be taken up and the trigger conditions. The trigger thresholds are automatically held at a level immediately above the noise level. For the timing of kinetic measurements a linear voltage ramp of adjustable rise time is available. A built‐in precision voltage source can be used for an overall calibration. The data system is operated by software written in assembly language. Data may be collected and processed in 1–8‐parameter listmode or 1–3‐parameter histogram mode. Functions are available for graphical color displays, numerical integration, multiparameter gating, and printing.
Flow cytometric detection of ornithine decarboxylase activity in epidermal cell subpopulations
Authors: Robertson Fredika M. , Gilmour Susan K. , Beavis Andrew J. , O'Connell Sean M. , Conney Alan H. , Huang Mon‐Tuan , Laskin Jeffrey D. , Hietala Oili A. , O'Brien Thomas G. 
Published: August 2005

AbstractUsing flow cytometry in combination with membrane permeabilization techniques to enhance binding of antibodies with immunoreactive protein within the cytoplasm, we have developed a method to examine the ornithine decarboxylase (ODC) activity present within subpopulations of epidermal cells following acute and chronic exposure to the phorbol ester tumor promoter 12‐0‐tetradecanoylphorbol‐13‐acetate (TPA). The method described has the sensitivity to detect basal levels of ODC as well as increases in ODC at early time points following treatment with TPA and has the additional advantage of allowing subpopulation identification and characterization.
Recovery of RNA from flow‐sorted fixed cells
Authors: Khochbin Saadi , Grunwald Didier , Pabion Michel , Lawrence Jean‐Jacques 
Published: August 2005

AbstractWe describe a method for the preparation of RNA from ethanol‐fixed cells, allowing analysis of the RNA from cells “frozen” in a given physiological state. This technique may have important applications in experiments which require prolonged cell manipulations beforre RNA preparation, such as investigations of cell‐cycle‐regulated gene exprression, which require the preparation of cells for cell‐cycle flow analysis, and even for long‐term cell sorting. It eliminates all the inconveniences associated with the use of fresh cells, and allows cell‐cycle biologists to couple flow cytometry methodology with the advancing techniques of molecular biology.
Flow cytometric measurement of rates of particle uptake from dilute suspensions by a ciliated protozoan
Authors: Lavin Daniel P. , Fredrickson A. G. , Srienc Freidrich 
Published: August 2005

AbstractFlow cytometry is used to measure rates of ingestion of particles from dilute monodisperse suspensions by the ciliate Tetrahymena pyriformis. The particles used are polystyrene microspheres containing a fluorescent dye. Measurements were made directly, that is, by determining the fluorescence intensities from microspheres ingested by cells in samples collected from the experimental feeding apparatus. The fact that fluorescence intensities from individual cells can be grouped into discrete classes based on the numbers of fluorescent particles associated with the cells makes it possible to calibrate the flow cytometer and convert fluorescence measurements into numbers of particles ingested by average cells. At low particle concentration or high ciliate concentration, ingestion data must be corrected for depletion of particles during the assay, and a method for doing this is described. Experiments at various ciliate concentrations show that ingestion rates are not affected by this concentration. The methods developed should allow measurements of rates of ingestion of particles from concentrated and polydisperse suspensions. For such measurements, nonfluorescent particles together with a fraction of fluorescent tracer particles would be used.
Alterations of growth fraction and DNA content in K562 cells by differentiating agents
Authors: Gorisse Marie‐Claude , Carpentier Yves , Joly Philippe , Comoe Léopold , Desoize Bernard 
Published: August 2005

AbstractThe growth fraction, estimated by the monoclonal antibody Ki‐67 labeling, and DNA content, assessed by ethidium bromide staining, were determined simultaneously in K562 leukemic cells by flow cytometry. A multiparametric analysis enabled the fraction of the cell by flow cytometry. A multiparametric analysis enabled the fraction of the cell population with G1, S, and G2 + M content in Ki‐67‐positive and Ki‐67‐negative cells to be evaluated.Butyric acid (BUT) was used as positive control. The fraction of Ki‐positive cells decreased with the BUT concentration, while the proportion of cells with G1 DNA content increased only in the Ki‐negative cells.Adriamycin, aclacinomycin A, and fagaronine induced differentiation, as assessed by benzidine staining and glycophorin A expression. These drugs decreased the fraction of Ki‐positive cells by more than 50% for both anthracyclines and by 25% for fagaronine. Following treatment, Ki‐negative cells displayed a G1, but also a G2 and a S DNA content in different proportions, indicating that induction of quiescent cells by differentiating agents is not a uniform process and is worthy of interest.
Detection of cells resistant to alkylating agents by flow cytometric analysis of DNA damage
Authors: Frankfurt Oskar S. , Seckinger Daniel , Sugarbaker Everett V. 
Published: August 2005

AbstractDNA damage was measured by flow cytometric analysis of cells sensitive and resistant to alkylating agents. Human ovarian carcinoma cell line A2780 and a subline which is 7 times more resistant to L‐phenylalanine mustard (L‐PAM) were treated with the drug, fixed, and stained with monoclonal antibody (MOAB) F7‐26 which detects single‐stranded regions in alkylated DNA. Mean fluorescent intensity was measured on a flow cytometer. Cells were heated before staining to amplify single‐strandedness in alkylated DNA. Significantly larger amount of MOAB was bound to DNA in sensitive than in resistant cells. Fluorescence increased by 80 channels per νg L‐PAM insensitive cells and only by 17 channels in resistant cells. Sensitive and resistant cells were treated with L‐PAM, mixed in different proportions, and stained with MOAB. Populations of sensitive and resistant cells were clearly separated on fluorescence histograms by more than a decade difference in fluorescence intensity. Presence of 2–5% resistant cells was detected among sensitive cells as a separate cell subset.We conclude that staining with MOAB F7‐26 can be used as an indicator of cell sensitivity or resistance to alkylating agents. Detection of minor subsets of resistant cells in heterogeneous populations by FCM analysis may be useful for monitoring emerging drug resistance.
Flow cytometric evaluation of nitro blue tetrazolium (NBT) reduction in human polymorphonuclear leukocytes
Authors: Fattorossi Andrea , Nisini Roberto , Le Moli Soccorsa , De Petrillo Guido , D'Amelio Raffaele 
Published: August 2005

AbstractOxidative metabolic burst of activated human polymorphonuclear leukocytes (PMN) is most commonly investigated in clinical practice by evaluating nitroblue tetrazolium (NBT) reduction at the single cell level. Reduced NBT precipitates where the redox reaction has taken place and can be visualized as PMN‐associated dark blue granules of formazan in light microscopy. Although widely used and not technically demanding, this method remains subjective and labor intensive, especially when large numbers of samples need to be investigated. We developed a new flow cytometry technique in which PMN membrane was rendered fluorescent by a short incubation with fluorescein‐conjugated Concanavalin A. PMN were then incubated with NBT and increasing doses of a suitable stimulus, such as phorbol myristate acetate (PMA). Formazan has a distinct peak of absorption at 520 nm that represents the peak of emission of fluorescein. As a consequence, formazan quenches the PMN‐associated fluorescence. Data show that a dose‐dependent reduction of fluorescence can be obtained using graded amounts of PMA in normal PMN cultures. PMN‐associated fluorescence remains unchanged in control patients with chronic granulomatous (CGD) disease, a disorder characterized by a selective impairment of PMN oxidative metabolism. Electronic cell size increases upon PMA incubation in normal PMN, irrespective of the presence of NBT. Conversely, forward light scatter intensity decreases in the presence, but not in the absence, of NBT indicating that the phenomenon is due to the capacity of formazan to absorb/scatter the incident light. The present method for easily detecting NBT reducing activity at single cell level by flow cytometry makes use of commonly available, inexpensive reagents and standard instrumentation. It could become a useful test for clinical purposes.
Personal computer interface and software for the ZONAX microscope attachment controller
Authors: Rigney David R. , Velyvis John H. , Wei Jeanne Y. 
Published: August 2005

AbstractWe describe the hardware and software of a general‐purpose interface that permits a personal computer (PC) running MS‐DOS to control cytometric devices, e.g., a scanning stage, shutters, and focus motor attached to a microscope. This note explains how to use the interface to convert the ZONAX microscope attachment controller from 8080‐based hardware control to PC‐based control.
Improved method for measuring intracellular Ca++ with fluo‐3
Authors: Rijkers Ger T. , Justement Louis B. , Griffioen Arjan W. , Cambier John C. 
Published: August 2005

AbstractThe accuracy of flow cytometric measurement of intracellular calcium with fluo‐3 is compromised by variation in basal fluorescence intensity due to heterogeneity in dye uptake or compartmentalization. We have loaded cells simultaneously with fluo‐3 and SNARF‐1. When SNARF‐1 fluorescence is collected at approximately 600 nm, its intensity does notchange upon cell activation. Furthermore, fluo‐3 and SNARF‐1 fluorescence signals exhibit a linear relationship. The ratio of fluo‐3 to SNARF‐1 eliminates a significant proportion of variation in fluorescence intensity caused by variation in fluo‐3 uptake and thus can be used as a sensitive parameter for measuring changes in [Ca2+]i.
Nuclear texture parameters as discriminant factors in cell cycle and drug sensitivity studies
Authors: Colomb Evelyne , Dussert Christophe , Martin Pierre‐Marie 
Published: August 2005

AbstractThe authors have recently shown that cell cycle characteristics of in situ cell populations can be determined using the SAMBA 200 cell image processor by computing 15 densitometric and texture parameters on each Feulgen‐stained nucleus and multiparametric analysis of data. The present paper displays the importance of chromatin pattern assessment and detection of conformational changes in DNA structure, based on nine nuclear texture parameters measured from the grey level cooccurrence and the run‐length section matrices.Reference files were constructed by merging respective reference files (G0/ G1, S, G2 and M) of MDA AG and MCF‐7, two mammary epithelial cell lines presenting different morphological aspects and hormone responses, these files were found to be valid in the reclassification of any mammary epithelial cell in culture with a diploid or near diploid pattern.Moreover, the authors demonstrate that chromatin texture changes, following direct interaction of chemotherapeutic drugs with DNA, may be assessed owing to nuclear texture parameters. Consecutive to daunomycin addition (0.5 μg/ml) and concomitant to the appearance of nuclear morphological alterations in MDA AG sensitive cells as viewed by microscopic observation, discriminant factorial analysis showed progressively increasing erroneous reclassification from 15 to 72 h of treatment. These experimental results were exploited with a kinetic mathematical model to quantify the daunomycin blocking effect: 20% in S phase and 80% in G2 phase. Interestingly, no textural change was observed on MDA A1 anthracycline resistant cells, indicating that these texture parameters may permit distinction of drug sensitive cells.This methodology (1) can be applied to test in vitro resistance‐reversal molecules, (2) may be extended to other therapeutic agents giving rise to conformational changes in DNA structure, and (3) can be applied to cytopunctions or imprints of tumor biopsies with diploid‐like DNA content to follow evolution of drug sensitivity or resistance during course of therapy.
Modalities of synthesis of Ki67 antigen during the stimulation of lymphocytes
Authors: Lopez F. , Belloc F. , Lacombe F. , Dumain P. , Reiffers J. , Bernard P. , Boisseau M. R. 
Published: August 2005

AbstractThe antibody Ki67 is currently used to evaluate the proliferative fraction of solid tumors and some hematological malignancies. We have used phytohemagglutinin (PHA)‐stimulated peripheral blood lymphocytes as a model to study the entry of quiescent cells into cell cycle and to follow their progress to the next cycle. Flow cytometric analysis of lymphocyte samples stained with the antibody Ki67 and a DNA marker has allowed us to follow the expression of Ki67 antigen (Ki67Ag) as a function of the position of the cells in the cell cycle. The use of drugs blocking the stimulated lymphocytes in different phases of the cell cycle permitted us to demonstrate that Ki67Ag expression started from the beginning of the first S phase. The level of Ki67Ag increased during S phase until mitosis, when its expression was maximal. After division, the cells in G1 phase showed a decrease in Ki67Ag expression (possibly corresponding to degradation) until they reentered S phase, when the level of Ki67Ag increased again. The results confirm that the expression of Ki67Ag is related to the proliferative state of the cells and suggest that it may be used to determine the proliferative cell fraction in hematopoietic tissues.
Aberrant Ki‐67 expression in normal bone marrow revealed by multiparameter flow cytometric analysis
Authors: Van Bockstaele Dirk R. , Lan Jar , Snoeck Hans‐W. , Korthout Marcel L. , De Bock Robrecht F. , Peetermans Marc E. 
Published: August 2005

AbstractKi‐67 is a commercially available monoclonal antibody that reacts with a nuclear antigen detectable in proliferating cells only. Since its first description, it has been widely used as a “universal” proliferation marker and few groups have questioned the validity of the initially described reactivity, although this was tested only on very restricted experimental models. We wanted to check its reactivity on normal bone marrow (BM) samples using a multiparameter flow cytometric analysis. Although we were able to reproduce the findings of Ki‐67 positivity on cultured and stimulated cells, we could not detect any convincing Ki‐67 positivity on nuclei of normal BM samples. These samples all had a noticeable proliferating compartment as evidenced by their DNA content. These data are in contrast with the data we obtained starting from stressed marrows and marrows cultured in the presence of hematopoietic growth factors, where we found a marked Ki‐67 positivity.This discrepancy suggests that bone marrow cells, growing and proliferating under steady‐state conditions and guided by natural control mechanisms, may lose their Ki‐67 expression upon exiting the progenitor compartment and entering the differentiating compartment.
Introduction of antibody into viable cells using electroporation
Authors: Berglund Deborah L. , Starkey Jean R. 
Published: August 2005

AbstractConditions for labelling an intracellular antigen, p21ras, using electroporation to introduce a fluorescent antibody, are described. Following labelling, cells were evaluated for p21ras associated fluorescence by flow cytometry. Electroporation, sorting, and cell handling parameters were varied to determine optimal conditions for cell viability. Cells were best held in serum containing growth medium both before and after electroporation, while antibody introduction during the electroporation phase was most efficient when carried out in a balanced saline solution. For maximum efficiency of antibody internalization, the antibody needed to be present during electroporation, and medium needed to be replaced several times in the first few hours after electroporation to ensure good cell survival.
DNA histogram debris theory and compensation
Authors: Bagwell C. Bruce , Mayo Sara W. , Whetstone Sherry D. , Hitchcox Shelly A. , Baker David R. , Herbert Donald J. , Weaver Donald L. , Jones Michael A. , Lovett Edmund J. 
Published: August 2005

AbstractIn this paper we describe a theory of DNA histogram debris generation and compensation that can be applied to paraffin‐embedded frozen tissue preparations. The theory predicts the distribution of fragments generated from single and multiple random sectioning of threedimensional ellipsoids representing nuclei. The fragment distribution is assumed to be a major component of the underlying debris in DNA histograms. A comparison of S‐phase fractions (SPF) from matched tissue prepared by frozen and formalin‐fixed paraffin‐embedded DNA methods demonstrates the usefulness of the theory.
Ratiometric measurement of intracellular pH in cultured human keratinocytes using carboxy‐SNARF‐1 and flow cytometry
Authors: Van Erp Piet E. J. , Jansen Monique J. J. M. , De Jongh Gijs J. , Boezeman Jan B. M. , Schalkwijk Joost 
Published: August 2005

AbstractIn this study we describe a method to measure intracellular pH in cultured human keratinocytes using flow cytometry. Keratinocytes pose a technical problem because the population is heterogeneous with respect to size and metabolic activity (nonspecific esterase activity), resulting in variability in dye uptake. In order to compensate for this, dyes were selected that change colour with pH. The ratio of fluorescence intensities at two wavelengths was recorded and used as a measure of intracellular pH by reference to the pH in the presence of the proton ionophore nigericin. However, methods published till now do not routinely combine the ratiometric technique and excitation with an argon ion laser set at 488 nm. Therefore we have tested the recently developed pH‐sensitive dye carboxyseminaphthorhodafluor‐1 (SNARF‐1) as a possible candidate for flow cytometric pH measurements and compared it with 2′,7′‐bis(carboxyethyl)‐5,6‐carboxyfluorescein (BCECF) and 2,3‐dicyanohydroquinone (DCH) with respect to emission spectra, resolution, range, and stability of cellular fluorescence. SNARF‐1 had a practical and stable excitation wavelength of 488 nm rather than UV, it offered the possibility of ratio‐metric measurements on the basis of a real emission shift, and had superior resolution for the pH range 7–8. With SNARF‐1 we found that keratinocytes cultured under low serum conditions (0.2%) contain a higher proportion of cells with relatively low intracellular pH compared to high serum cultures (6%). Furthermore, pH changes were followed by changes in relative DNA content. These findings suggest that intracellular pH can be an early functional proliferation marker for human keratinocytes.
Use of a photolabeling technique to identify nonviable cells in fixed homologous or heterologous cell populations
Authors: Riedy M. C. , Muirhead K. A. , Jensen C. P. , Stewart C. C. 
Published: August 2005

AbstractFlow cytometric determination of viable versus nonviable cells in fixed samples can be accomplished by utilizing the irreversible binding of photoactivated ethidium monoazide (EMA). EMA is a positively charged molecule which is excluded by cells with intact membranes (viable cells), included by cells with damaged membranes, and can be photochemically crosslinked to nucleic acids using visible light. EMA fluorescence can be excited using a standard argon laser operating at 488 nm and is able to be distinguished from fluorescein and phycoerythrin, Fixation is important when analyzing cells from a potentially infectious origin. EMA is photochemically crosslinked and therefore unable to leak out of cells when removed from the extracellular media, unlike propidium iodide (PI) or other viability stains, which were heretofore commonly used. We demonstrate the usefulness of EMA in combination with fluoresceinated and phycoerythrin labeled monoclonal antibodies in immunophenotyping. The photoaffinity labeling technique allows for a quick and efficient means of identifying nonviable cells which cannot be distinguished on the basis of light‐scattering properties.