bluuebunny/crossref_metadata_2025_split huggingface dataset:
Digital morphonuclear analyses of sensitive versus resistant neoplastic cells to vinca‐alkaloid, alkylating, and intercalating drugs
Authors: Pauwels Olivier , Kiss Robert 
Published: August 2005

AbstractWe tested 12 resistant cell lines in vitro in order to evaluate common morphonuclear characteristics induced by various cytotoxic drugs on cell lines of different origins. We used the MXT mouse mammary cancer and the neoplastic J82 and T24 human bladder cell lines, whose variants are either sensitive or resistant to a vinca alkaloid derivative (Navelbine, NVB), to an investigational alkylating agent (PE1001), and to Adriamycin (ADR). We tested cell population variants resistant to NVB + PE1001 + ADR. The level of chemoresistance was evaluated by a colorimetric assay assessing the 50% concentration‐induced inhibition of cellular growth (IC50) brought about by each drug on the growth of each cell variant under study. We show that resistant neoplastic cell nuclei present common morphonuclear characteristics, independent of cell origin (neoplastic mouse mammary versus human bladder cells) and the drug used (vinca alkaloid, alkylating, and intercalating derivatives). Our results further indicate that the phenotype of resistant versus sensitive cells corresponds to cell nuclei populations with smaller nuclei and less nuclear DNA content and, as a consequence, a chromatin texture showing large pale areas with some hyperchromatic clumps.
Washless double staining of unfixed nuclei for flow cytometric analysis of DNA and a nuclear antigen (Ki‐67 or bromodeoxyuridine)
Authors: Larsen Jørgen K. , Christensen Ib Jarle , Christiansen Jette , Mortensen Børge Thing 
Published: August 2005

AbstractWashless methods for double staining of nuclear antigen and DNA in unfixed nuclei were compared with established methods for staining of fixed cells. The methods were tested on phytohemagglutinin (PHA)‐stimulated normal human blood lymphocytes for the double staining of (1) Ki‐67 antigen and DNA and (2) bromodeoxyuridine (BrdUrd) and DNA, in continuously BrdUrd‐labeled cells. With respect to the discrimination between antigen‐positive and ‐negative subpopulations, there was no statistically significant differences between the results from direct (Ki‐67) or indirect, (Ki‐67 or BrdUrd) washless staining of unfixed nuclei and the results from staining of fixed cells. Washless staining of unfixed nuclei was found to be rapid and simple and resulted in greater precision of the DNA analysis and in less aggregation and loss of cells.
Simple delay monitor for droplet sorters
Authors: De Grooth Bart G. , Doornbos Richard M. P. , Van Der Werf Kees O. , Greve Jan 
Published: August 2005

AbstractWe have constructed a simple device by which the optimal delay time between optical measurement of a cell and the application of the droplet charging pulse can be determined directly in a flow sorter. The device consists of a stainless steel chamber in which the sorted droplets are collected. In the collection chamber the collected droplets run through a capillary where a continuous fluorescence measurement is made. With a sample of fluorescent particles, the delay time is optimal when the measured fluorescence is maximal. The measuring volume is always filled with the last droplets sorted (about 3,000). With this device, the setting of the delay time can be done in a few seconds without the need for microscopical verification.The fluorescence in the collection chamber is excited and detected via optical fibers using about 10% of the light of the existing laser from the flow cytometer and an extra photomultiplier.
Interlaboratory study of cellular fluorescence intensity measurements with fluorescein‐labeled microbead standards
Authors: Vogt Robert F. , Cross G. David , Phillips Donald L. , Henderson L. Omar , Hannon W. Harry 
Published: August 2005

AbstractTo determine the precision of cellular fluorescence intensity (FI) measurements derived from labeled microbead standards, FI results were compared from 43 different flow cytometers in 34 laboratories. All laboratories analyzed prepared aliquots of fluoresceinated calf thymocyte nuclei (Fluorotrol), human lymphocytes stained with fluoresceinated anti‐CD4 antibody, and fluoresceinated microbeads used as both internal and external standards. Measurements were conducted by most laboratories on the third and fourth days after sample preparation. Results for percent of events within the gates and the histograms returned by participants indicated that the samples had remained stable and that gated populations had been properly identified. All standard curves showed strong linearity, and the pooled results from all standards produced a best‐fit curve that was in close agreement with the assigned values. Nonetheless, results for cellular FI were highly variable, with CVs of 20–34%. Agreement within lab/instrument was much better, with CVs ranging from 3.0 to 9.9%. The overall variability was not obviously attributable to differences in the types of cytometer, nor could it be explained by attributes of the standard curves or any other single variable examined. However, the application of a corrective factor based on FI results for Fluorotrol allowed a two‐fold improvement in the precision of FI measurements on CD4‐stained lymphocytes, with an overall CV of 11%. Uncharacterized differences in the operating conditions of flow cytometers can influence cellular FI measurements, but consistent results can be obtained if a stained cellular calibrator is analyzed in addition to the proper microbead standards.
Two‐ and three‐color immunofluorescence using aminocoumarin, fluorescein, and phycoerythrin‐labelled antibodies and single laser flow cytometry
Authors: Delia Domenico , Martinez Erundina , Fontanella Enrico , Aiello Antonella 
Published: August 2005

AbstractAntibodies coupled to 7‐aminocumarin (AMCA) emit a bright blue fluorescence under ultraviolet (UV) excitation and are therefore ideal for three‐color immunofluorescence (IF) with fluorescein (FITC) and phycoerythrin (PE) labeled reagents; however, due to the different absorption spectra, the use of these fluorophores for multicolor flow‐cytometric analysis requires a double light excitation source (e.g., two‐laser system).We report a strategy which uses a single argon‐ion laser to simultaneously excite AMCA, FITC, and PE, thus allowing the flow cytometric analysis of three immunological parameters. When the UV‐visible argon‐ion laser is fitted with an appropriate set of mirrors, the 351.1–363.8 nm (UV) and 488 nm wavelengths (accounting for 80 mW and 520 mW, respectively) are simultaneously generated; these lines can then be exactly focused on the same observation point by an achromatic cylindrical lens.A number of comparative analysis were performed with this instrumental set up to verify the sensitivity of AMCA IF and its possible application for multicolor immunophenotypic evaluation of blood cell subsets.When AMCA‐ and FITC‐labeled anti‐mouse Ig antibodies were assessed for their ability to detect limiting amounts of mouse monoclonal antibody bound to cells, the former was less sensitive than the latter. A number of factors, including differences in excitation energy (80 mW for AMCA and 520 mw for FITC) and extinction coefficients (1.9 × 104 for AMCA and 6 × 104 for FITC) could explain this result.Single‐color IF analysis of blood cells stained with monoclonal antibodies specific for T‐cells (CD3, CD4, and CD8) and B‐cells (CD19) gave, for each marker, similar results, regardless of the fluorochrome used; similarly, two‐color IF using AMCA/FITC, AMCA/PE, and FITC/ PE combinations provided comparable results. AMCA fluorescence was minimally detected in the green (requiring only 5% color compensation) but not in the red fluorescence channel. Triple‐color detection of CD3, CD4 (or CD8), and CD 19 positive blood cell subsets was also achieved.Our data indicate that multiple‐color IF analysis of blood T‐ and B‐cell subsets can be performed by flow cytometry and single‐laser dual‐wavelength excitation using antibodies labeled with AMCA, FITC, and PE. Improvements of the fluorescent reagents and of the optical assembly of the instrument may be required to further increase the resolution of AMCA IF.
Imaging cytometry by multiparameter fluorescence
Authors: Galbraith William , Wagner Marc C. E. , Chao Jean , Abaza Mohammed , Ernst Lauren A. , Nederlof Michel A. , Hartsock Robert J. , Taylor D. Lansing , Waggoner Alan S. 
Published: August 2005

AbstractA system is described for performing multicolor fluorescence image cytometry of cell preparations. After the setting up stage, the operation is automatic: the microscope fields are found and focused; then images are acquired for each fluorophore, corrected and analyzed, without any operator interaction. Human peripheral blood lymphocytes on microscope slides were used as a test system. In these experiments, three fluorescent antibodies were used to identify lymphocyte sub‐populations, and a DNA content probe was used to identify all nucleated cells. The cell subset percentages determined by image cytometry were comparable to percentages obtained when cells from the same preparation were analyzed by flow cytometry. Multicolor fluorescence imaging cytometry can potentially be extended to the analysis of cells in smears, fine needle biopsies, imprints, and tissue sections.
Time window analysis and sorting
Authors: Dunne John F. 
Published: August 2005

AbstractFlow cytometric hardware and procedures were developed to continuously analyse and to sort a particular time window in a kinetic response. The technique uses balanced air pressure to drive a stimulus‐bead mixture from a vial to a t‐junction where it mixes passively with cells. The t‐junction is distanced from the flow cell and air pressure regulated so that the stimulation occurs at a fixed and adjustable time before the cells are interrogated by the laser beam. Practical applications of the device demonstrate utility with cells whose reponses are seen in seconds or minutes. The device is easily implemented on any sorting flow cytometer.
Tumor cytokinetic effects of acute starvation versus polyamine depletion in tumor‐bearing mice
Authors: Westin Thomas , Gustafsson Bertil , Hellander Karin , Reinholdtsen Lola , Tibell Lena , Lundholm Kent , Edström Staffan 
Published: August 2005

AbstractPrevious investigations in our laboratory have demonstrated that both acute host starvation and polyamine depletion by means of the irrevesible ODC‐inhibitor (ODC = ornithine‐decarboxylase) fluoro‐methylornithine (DFMO) lead to pronounced growth retardation of rapidly proliferating tumors. The aim of this investigation was to elucidate how these different interventions affect cell kinetics and cell cycle phases in vivo. Adult nongrowing mice (C57Bl/J) bearing a poorly differentiated rapidly growing methylcholanthrene induced sarcoma were used. Combined measurements of bromodeoxyuridine incorporation into DNA and flow cytometric techniques were used.Starvation and DFMO treatment resulted in a prolonged cell cycle transit compared to freely fed animals. Tumor cells from DFMO‐treated mice demonstrated an increased time for DNA synthesis and a relatively larger accumulation of cells in the G2M phase, whereas tumor cells from starved animals were accumulated in the G0G1 phase. The fractional cell loss of tumor cell during proliferation was calculated to be around 18% higher in DFMO‐treated animals compared to starved and freely fed tumor‐bearing mice.This study demonstrates that different mechanisms are involved in tumor growth supression from substrate deficiency (starvation) and from inhibition of polyamine synthesis.
Characterization of effector‐target conjugates for cloned human natural killer and human lymphokine activated killer cells by flow cytometry
Authors: Callewaert Denis M. , Radcliff Gilbert , Waite Ross , LeFevre Janet , Poulik M. Dave 
Published: August 2005

AbstractThe present studies demonstrate that the intracellular fluorochromes calcein and hydroethidine can be used for quantification of effector‐target conjugates involving cloned human natural killer (NK) or interleukin‐2 (IL‐2) activated human lymphokine activated killer (LAK) cells by dual color flow cytometry without potential artifacts that might result from extensive modification of effector and/or target cell membranes. Cloned NK cells and LAK cells form conjugates with cultured cell lines regardless of susceptibility to lysis. The strength of the interactions in these conjugates was investigated using a variable speed vortexer. Even relatively gentle vortexing disrupted most conjugates involving fresh human peripheral blood lymphocytes (PBL) but only about one‐fourth of conjugates between K‐562 cells and human PBL that had been cultured with or without IL‐2 by this treatment.The rate of conjugate formation for LAK cells was determined to be about 3 times faster than for cloned NK cells, and both rates are considerably faster than the reported rate of formation of cytotoxic T lymphocyte (CTL) target conjugates. The differences in the rate of conjugate formation are apparently not related to target cell specificity, since LAK cells form conjugates with susceptible and resistant cell lines at comparable rates. When effector‐target conjugates are incubated at 37°C in the absence of calcium—thereby precluding lysis—the percentage of conjugated LAK or cloned NK cells decreases logarithmically with time. These results suggest that an initial equilibrium between free and conjugated lymphocytes gradually shifts in favor of unconjugated cells.
Assessment of radiation‐induced DNA strand breaks and their repair in unlabeled cells
Authors: Affentranger Maja Item , Burkart Werner 
Published: August 2005

AbstractRadiation induced damage, i.e., the induction of DNA strand breaks, was studied on the level of single, unlabeled cells. DNA strand breaks were determined by direct partial alkaline unwinding in intact cell nuclei followed by staining with acridine orange, a development of a proposal: first described by B. Rydberg (Int J Radiat Biol 46:521–527, 1984). The ratio of green fluorescence (doublestranded DNA) to red fluorescence (single‐stranded DNA) in single cells was taken as a measure of DNA strand breaks. CHO‐K1 and M3‐1 cells irradiated with X‐rays show a dose dependent induction of DNA strand breaks. Incubation at 37°C after irradiation leads to repair of breaks. A repair halflife of about 10–11 min can be determined.Cell cycle specific differences in the induction of DNA strand breaks or repair behavior are not detectable at the resolution achieved so far.This new method offers two major advantages: the resolution of DNA damage and repair on the level of single cells and no need for labeling, thereby allowing for DNA damage and repair to be assessed in biopsy material from tumor patients.
Improved staining method for the simultaneous flow cytofluorometric analysis of DNA content, S‐phase fraction, and surface phenotype using single laser instrumentation
Authors: Toba Ken , Winton Elliott F. , Bray Robert A. 
Published: August 2005

AbstractWe have developed an improved technique for triple staining that permits the simultaneous flow cytofluorometric analysis of cell surface antigens, bromodeoxyuridine incorporation into DNA, and DNA quantification using 7‐amino‐actinomycin D. PHA‐activated human peripheral blood lymphocytes were incubated with bromodeoxyuridine and stained for cell surface phenotype with phycoerythrin‐labeled monoclonal antibodies. Stained cells were fixed serially with 1% paraformaldehyde and 45% ethanol. Fixed cells were sequentially stained with an anti‐BrdUrd monoclonal antibody followed by a FITC‐conjugated goat anti‐mouse antibody and incubated with 7‐amino‐actinomycin D. Hypotonic buffer was employed for all procedures after fixation. Stained‐fixed cells were analyzed by flow cytofluorometry for simultaneous green (525 nm), orange (570 nm), and red (> 650 nm) fluorescence. Utilizing this staining technique, we were able to analyze simultaneously cell phenotype, DNA synthesis, and total cellular DNA content with single laser excitation.
In vivo measure of average bacterial cell size from a polarized light scattering function
Authors: Bronk Burt V. , Van De Merwe Willem P. , Stanley Marc 
Published: August 2005

AbstractA particular combination of elements of the Mueller matrix for scattering of polarized light given by
(S34 + S14)/(S11 + S13)  (S34/S11)‡
is measured vs angle at a wavelength of 633 nm for randomly oriented suspensions of several species of bacteria in different stages of growth. (This combination of elements is dominated in the present measurements by the behavior of the normalized S34 matrix element, as is indicated by the notation defined on the right side of the equation.) The resulting graph in each case shows an oscillating function of angle. This function is compressed toward smaller angles when the bacteria are in the exponential phase of growth in comparison with results for a suspension of the same bacteria in the stationary (starving‐smaller cells) phase of growth. Microscopic measurements were made to determine, for each case, the average dimensions of the bacterial population. Graphs were then plotted of the peak positions from the Mueller matrix function plots vs either cell length or cell diameter. The function was shown to be strongly correlated with cell diameter under the conditions of this experiment and poorly correlated with cell length. The measurements were shown to have a sensitivity to changes in average diameter of about 20 nm.
Measuring percent lymphocytes by flow cytometry to calculate absolute lymphocyte subset counts for HIV + specimens
Authors: Gale Howard B. , Henry Keith 
Published: August 2005

AbstractA comparison was made of lymphocyte percentages from an automated hematology analyzer (ELT 15) vs. a fluorescence flow cytometer (Cytofluorograf). The hematology values were consistently higher than the flow (by greater than 10% for 13 of 50 HIV + specimens). The findings were similar to three other pairings: H*1 vs. Cytofluorograf, Cell Dyn 3000 vs. Cytofluorograf, and Cell Dyn 3000 vs. EPICS Profile. In another comparison manual percent lymphocytes matched much better with flow values. Factors of sample preparation and instrument analysis as they relate to nonrandom cell loss and lymphocyte resolution are examined.
Dead cell discrimination with 7‐amino‐actinomcin D in combination with dual color immunofluorescence in single laser flow cytometry
Authors: Schmid Ingrid , Krall Wanda J. , Uittenbogaart Christel H. , Braun Jonathan , Giorgi Janis V. 
Published: August 2005

AbstractIdentification of nonviable cells in immunofluorescently stained cell populations is essential for obtaining accurate data. Fluorescent non‐vital DNA dyes, particularly propidium iodide (PI), have been used routinely in flow cytometry for discrimination of dead cells from viable cells on the basis of fluorescence. We describe here the use of an alternative DNA dye, 7‐amino‐actinomycin D (7‐AAD), which can replace PI for the exclusion of nonviable cells. As an example, we present in this paper the utilization of 7‐AAD on various leukemic cell lines for dead cell exclusion whenever the viable cell population could not be discriminated reliably from nonviable cells on the light scatter histogram; 7‐AAD is suitable for dead cell discrimination in lengthy experiments because it is efficiently excluded by intact cells and has a high DNA binding constant. In addition, the dye is valuable in combination with phycoerythrin (PE)‐fluorescence dual‐color flow cytometry on a single argon laser instrument, since its emission in the far red can easily be separated from the emission of PE; 7‐AAD was used on fluoresceinisothiocyanate (FITC) and PE surface‐labeled human thymocytes for characterization of the dying subpopulation of cells which is undergoing programmed cell death. In this heterogeneous cell preparation, the spectral properties of the dye permitted the classification of viable and nonviable cell subpopulations by multiparameter analysis.
Evaluation of breast carcinoma chemosensitivity by flow cytometric DNA analysis and computer assisted image analysis
Authors: Briffod Marianne , Spyratos Frédérique , Hacène Kamel , Tubiana‐Hulin Michèle , Pallud Claude , Gilles Frédéric , Rouëssé Jacques 
Published: August 2005

AbstractFlow cytometric (FCM) DNA and SPhase (S%) analyses were compared to computerized image analysis (SAMBA 2005) in 27 breast carcinomas (T3, N0‐N1, M0) treated by 3 cycles of preoperative Adriamycin, vincristine, cyclophosphamide, methotrexate, 5‐fluorouracil (AVCMF) chemotherapy (CT). Twelve carcinomas had shown objective regression and 15 no, regression. Samples studied were obtained by sequential fineneedle cytopunctures. Comparing DNA profiles obtained by both methods before and after the first cycle, it appears that tumors can be divided into 3 groups. In the first group (10 cases), no changes were observed after the first cycle of CT. These tumors before treatment had either single DNA peak without cells in S% and G2M or a major peak with a small S% and G2M peak. The second group (9 cases) showed some changes in DNA profiles with an increased G2M peak but no additional values; these tumors before treatment had a small S% and a G2M peak. In the third group (8 case), before treatment, all were non‐diploid with high S% and high G2M. After the first cycle, all showed obvious changes in DNA profiles with a decrease of the G0/G1 peak and an increased S% and G2M with dispersed additional values along the scale in (G2M) × 2 and (G2M) × 4 regions. When changes were compared to tumor regression in the 1st and 2nd groups, 1/10 and 3/9 cases, respectively, were evaluated as objective regression. In the third group, all had objective regression (p < 0.001). In most cases, a good correlation was observed with both methods. Nevertheless, peak position definition was better with FCM than with SAMBA, but SAMBA permits a better estimation of changes in DNA patterns induced by CT. Furthermore, SAMBA gives information on changes in other morphometric parameters, such as nuclear area that significantly increased in chemosensitive cells (p = 0.003), and in chromatin textural parameters. Therefore, FCM and SAMBA give additional information on the early effect of CT on primary tumor cells and could help to select responsive tumors.
Analyzing multivariate flow cytometric data in aquatic sciences
Authors: Demers Serge , Kim Junhyong , Legendre Pierre , Legendre Louis 
Published: August 2005

AbstractFlow cytometry has recently been introduced in aquatic ecology. Its unique feature is to measure several optical characteristics simultaneously on a large number of cells. Until now, these data have generally been analyzed in simple ways, e. g., frequency histograms and bivariate scatter diagrams, so that the multivariate potential of the data has not been fully exploited. This paper presents a way of answering ecologically meaningful questions, using the multivariate characteristics of the data. In order to do so, the multivariate data are reduced to a small number of classes by clustering, which reduces the data to a categorical variable. Multivariate pairwise comparisons can then be performed among samples using these new data vectors. The test case presented in the paper forms a time series of observations from which the new method enables us to study on the temporal evolution of cell types.