bluuebunny/crossref_metadata_2025_split huggingface dataset:
Differentiation of HL‐60 promyelocytic leukemia cells monitored by flow cytometric measurement of nitro blue tetrazolium (NBT) reduction
Authors: Blair Owen C. , Carbone Rocco , Sartorelli Alan C. 
Published: August 2005

AbstractReduction of nitro blue tetrazolium (NBT) to insoluble blue formazan granules occurs during the stimulus‐induced respiratory burst of mature granulocytes and is routinely used as an indicator of the extent of granulocytic differentiation of HL‐60 acute promyelocytic leukemia cells. In the present study, the differentiation of HL‐60 leukemia cells induced by dimethylsulfoxide (DMSO) or retinoic acid was monitored by flow cytometric (FCM) measurement of forward and 90° light scatter of NBT treated cells. Two‐parameter correlated analysis permitted a distinction between cells with increased forward and decreased 90° light scatter (NBT−), and cells with decreased forward and increased 90° light scatter (NBT+). Fixation of NBT treated cells with 1% paraformaldehyde facilitated flow cytometric analysis, and allowed differences in NBT reduction to be quantitated. DMSO‐induced cells expressed an all‐or‐none reduction of NBT to formazan, compared with retinoic acid treated cells that exhibited a graded response. Three parameter flow cytometric analysis of HL‐60 leukemia cells stained with propidium iodide in combination with NBT allowed the determination of the cell cycle distribution of NBT‐treated cells.
A convenient on‐line device for reagent addition, sample mixing, and temperature control of cell suspensisons in flow cytometry
Authors: Omann Geneva M. , Coppersmith Ward , Finney David A. , Sklar Larry A. 
Published: August 2005

AbstractWe describe a simple and inexpensive device which permits the addition of up to three different solutions into a cell suspension which is on‐line in a flow cytometer. The mixing chamber houses a disposable plastic cuvette stirred with a magnetic stirrer. The sample chamber is attached to a circulating water bath, hence accurate temperature control is achieved.Because the system is prepressurized and the sample line is very short, the delay time‐between the point of sample modification and the point of analysis is reduced to a few seconds. Thus reagents may be added rapidly, and kinetic measurements of high temporal resolution are possible. Because the temperature of the sample chamber is regulated, binding can be observed over longer time periods than was previously possible. We demonstrate the usefulness of this device in determining the binding of fluoresceinated hexapeptide to human neutrophils under conditions where the stimulus is infused into the cell suspension while on‐line in the cytometer.
DNase I sensitivity of nuclear DNA measured by flow cytometry
Authors: Roti Roti Joseph L. , Wright William D. , Higashikubo Ryuji , Dethlefsen Lyle A. 
Published: August 2005

AbstractThe DNase I digestion kinetics of DNA in isolated nuclei (from HeLa or murine mammary carcinoma, 67 cells) were assayed flow cytometrically by measuring the changes in ethidium bromide (EtBr) fluorescence following various digestion time intervals. The DNase I digestion curve was characterized by an initial 25–30% increase in fluorescence upon addition of the enzyme, a rapid reduction in fluorescence to approximately 50–55% in 30 minutes, and a limit digest of 45–50% beyond 45 minutes.Throughout digestion, the DNA histogram retained its characteristic bimodal shape, showing that histogram rearrangement was not responsible for the changes in EtBr fluorescence. Irradiation with 5 ± 106 rads (137Cs‐γ‐rays) or exposure to 50 mM EDTA caused an increase in EtBr fluorescence similar to that caused by DNase I, suggesting that DNA nicking and/or chromatin loosening were responsible for this increase. Residual DNA assayed by the solubilization of 14C‐TdR (thymidine)‐labeled DNA indicated a similar kinetic pattern without the initial increase. However, at the limit digest, the fraction of DNA remaining trichloroacetic acid (TCA) insoluble (10%) was smaller than that measured by loss of EtBr fluorescence (50% of initial, 40% of maximum). Part of this difference was due to the presence of TCA soluble DNA trapped within the nuclear matrix (15–20%). This trapped DNA was released when the digested nuclei were exposed to 0.5–1.0 M NaCl just prior to EtBr staining.Exposure of HeLa cells to three agents that are believed to cause changes in chromatin structure resulted in alterations in the DNase I digestion kinetics measured flow cytometrically. N‐butyrate exposure resulted in an increased rate of digestion. In contrast, heat shock and hypertonic shock resulted in a reduced rate of digestion.The proliferating status of the cells was reflected in the DNase I sensitivity of nuclear DNA. Nuclei from quiescent 67 cells showed a 22% reduction in EtBr fluorescence after 10 minutes of digestion, whereas nuclei from proliferating 67 cells showed a 26% loss. Additionally, the initial increase in fluorescence intensity was greater for quiescent cells, possibly reflecting a greater degree of chromatin loosening. Thus, the flow cytometric assay of DNase I sensitivity has been demonstrated to be a useful probe for chromatin structure.
Flow cytometric analysis and sorting of human endometrial cells after immunocytochemical labeling for cytokeratin using a monoclonal antibody
Authors: Oud Peter S. , Henderik Jos B. J. , Beck Hans L. M. , Veldhuizen José A. M. , Peter Vooijs G. , Herman Chester J. , Ramaekers Frans C. S. 
Published: August 2005

AbstractEndometrial cells in suspension were stained with propidium iodide and a monoclonal antibody against a cytokeratin intermediate filament protein specific for glandular and columnar cells (RGE 53). In this way columnar epithelial cells of the normal endometrium and of adenocarcinomas can be distinguished and separated by flow cytometry from non‐epithelial cells (fibroblasts and inflammatory cells) and squamous epithelial cells, all of which are negative for RGE 53. This makes it possible to analyse and also sort pure fractions of this particular tissue type for further studies. The use of propidium iodide allows simultaneous DNA flow cytometry of these columnar epithelial cells. Therefore, the use of antibodies to cytokeratin in combination with propidium iodide can be of help in analyzing and sorting pure fractions of both normal and malignant cells. This allows a more refined examination of complex cell mixtures using flow cytometry.
Flow cytometric determination of DNA content in malignant and benign bone tumours
Authors: Heliö H. , Karaharju E. , Nordling S. 
Published: August 2005

AbstractThe nuclear DNA content of 38 malignant and 25 benign bone tumours was measured by flow cytometry. The specimens were taken either from biopsies or from surgical specimens. Seventeen of 26 primary malignant bone tumours were aneuploid, 15 had a single aneuploid DNA content, and 2 had a biclonal abnormality. Thirteen of 15 osteosarcomas were aneuploid, but only 2 of 6 chondrosarcomas showed an aneuploid DNA content. Six of 12 metastatic malignant bone tumours were also aneuploid. All 25 benign tumours had a diploid DNA content. Cell cycle analysis showed that the proportion of cells in S‐ and G2M‐phases was higher in the malignant compared to benign tumours, indicating a higher proliferative activity. The increase was statistically significant (p < 0.05) both in diploid and in aneuploid tumours. Among five tumours studied after chemotherapy, four displayed a marked hyperdiploid abnormality. Aneuploidy and high proliferative activity both were highly associated with malignant bone tumours, suggesting that DNA flow cytometry may be an adjunct in the assessment of malignancy of bone tumours.
Flow cytometric enumeration of absolute lymphocyte number in peripheral blood using two parameters of light scatter
Authors: Smart Y. C. , Cox J. , Murphy B. , Enno A. , Burton R. C. 
Published: August 2005

AbstractA method was developed to measure the absolute lymphocyte count (ALC) of whole blood using the Spectrum III automated flow cytometer. Ninety‐nine samples of human peripheral blood were analysed on the Spectrum and the Coulter Counter S Plus II, to allow for comparison of the two machines. Regression analysis was used to test the extent of agreement between the sets of measurements on the two machines. The results demonstrated that the slope of the regression line was not significantly different from one, indicating a high level of correlation between Spectrum and Coulter ALC's. However, the mean difference between Coulter and Spectrum ALC's was not equal to zero, with the Spectrum giving counts approximately 10% lower than those of the Coulter machine. This is attributed to the different ways by which the two machines define a lymphocyte, the Spectrum III by two parameters of light scatter and the Coulter S Plus II by the single parameter of cell volume.
Fluorescence studies of Hoechst 33342 with supercoiled and relaxed plasmid pBR322 DNA
Authors: Sandhu Laurie Cochran , Warters Raymond L. , Dethlefsen Lyle A. 
Published: August 2005

AbstractThe fluorescence properties of Hoechst 33342 (HO 33342) were examined with plasmid pBR322 in the supercoiled (Form I) or relaxed covalently closed circular (Form I°) conformation in order to determine whether qualitative or quantitative differences in fluorescence properties might provide an assay for topological states of DNA. It was found that HO 33342 exhibited a 30% greater fluorescence intensity with Form I pBR322, independent of the dye or DNA concentration. As the dye to DNA ratio was increased, a red shift of approximately 8 nm was observed for HO 33342 complexed with Form I or Form I°. The red shift in fluorescence emission occurred at higher HO 33342 concentrations with Form I vs. Form I° DNA; however, when Form I and Form I° were mixed in various proportions, neither the fluorescent intensity differences nor the HO 33342 concentration at which the wavelength shift occurred could be used to quantitate the relative proportions of topological states present. These results suggest that although the fluorescence properties of HO 33342 complexed with Form I DNA are different than those of HO 33342 complexed with Form I° DNA, the fluorescence assay is not sufficiently sensitive to quantitatively discriminate among a mixture of DNA in various topological states.
A method for simultaneous nuclear immunofluorescence and DNA content quantitation using monoclonal antibodies and flow cytometry
Authors: Clevenger Charles V. , Bauer Kenneth D. , Epstein Alan L. 
Published: August 2005

AbstractA preparative technique for the two‐parameter flow cytometric study of nuclear antigen expression is reported. This method employs a brief sequential treatment of cells at 4°C first with 0.5% paraformaldehyde and second with 0.1% Triton X‐100 in phosphate‐buffered saline followed by cellular staining with indirect immunofluorescence and propidium iodide. Using this technique, cellular morphology is preserved, cell clumping is minimized, and high‐quality indirect immunofluorescence and DNA staining are obtained with a minimum of nonspecific labeling. Utilizing nuclear antigen‐specific monoclonal antibodies in conjunction with this technique, the cell‐cycle phase‐dependent expression of such antigens is examined. From these data, the utility of two‐parameter flow cytometry in the identification and quantification of cell‐cycle‐dependent modulation of nuclear antigens is discussed.
Flow cytometric evaluation of cell dispersion from human head and neck tumors
Authors: Th. Bijman J. , Th. Wagener D. J. , van Rennes H. , Wessels J. M. C. , van den Broek P. 
Published: August 2005

AbstractThe preparation of single‐cell suspensions from 25 human head and neck tumors is described. Dispersal was performed overnight at 4°C under slight agitation of the tissue suspensions using various combinations of enzymes and additives. The cell suspensions were examined for number of cells released, viability, amount of debris, and DNA distribution by means of flow cytometry (FCM).It was shown that both trypsin/dithioerythritol (TD) and collagenase/DNase (CDse) were of value in dispersing single cells from tumor tissue. In contrast to CDse, incubation with TD appeared to be cytolytic to normal lymphocytes. In a number of cases, DNA‐FCM revealed ploidy abnormalities in a TD‐suspension, which were not discernible in the concurrent CDse‐suspension. Cell culture of primary cell suspensions corroborated the reliability of the DNA‐FCM measurements. Pretreatment with CDse improved tumor disaggregation by TD and indicated a different dispersal capacity.Addition of Ca2+ and Mg2+ ions to the dispersal mixtures and preincubation of tumor slices in complete medium for 1 day before initiation of cell dispersion influenced favorably the quality of the cell suspension.
Detection of minimal disease in acute leukemia using flow cytometry: Studies in a rat model for human acute leukemia
Authors: Martens Anton C. M. , Hagenbeek Anton 
Published: August 2005

AbstractThe study was made using a rat model for human acute myelocytic leukemia (BNML), which shows striking similarities with human acute myelocytic leukemia (AML). A monoclonal antibody (MCA‐Rm124), raised against BNML cells, allowed the recognition of the leukemic cell fraction. The discriminative capacity of the monoclonal antibody is based on differences in labeling intensities between normal and leukemic cells. After i.v. cellular transfer of leukemia, the growth of the leukemic cell population in the bone marrow, the liver, and the spleen was monitored using MCA‐Rm124 and flow cytometry. For the bone marrow and the liver, a clonogenic assay for leukemic cells was used to quantify the cell content in these organs. A good correlation was found between the bioassay‐derived data and the flow‐cytometry‐derived data. The doubling times of the leukemic cell population were not equal for the two organs studied, indicating that a number of different processes contribute to the net cell production per organ.Apart from their application in the detection of residual leukemia, monoclonal antibodies might be employed in the analysis of the growth kinetics of the “invisible” leukemic cell population.
Active analog pipeline delays for high signal rates in multistation flow cytometers
Authors: Parson J. D. , Hiebert R. D. , Martin J. C. 
Published: August 2005

AbstractFlow cytometers with sequential measuring stations require alignment of event timing information to assure that multiparameter data for each cell are properly correlated. Information from the first detector must be delayed until signals are produced at the last detector, with adjustments to align them in time for simultaneous digitization after the last measurement is completed. By using an analog “pipeline” delay, the deadtime between the first and last measuring stations can be minimized. The described device is capable of acquiring and propagating many signals within the delay period, with good fidelity at high signal rates. An integrated circuit charge‐coupled device (CCD), which is an analog shift register, is shown to be useful as a signal delay in the time range from 22 μs to several milliseconds.
The optimal application of forward and ninety‐degree light scatter in flow cytometry for the gating of mononuclear cells
Authors: Thompson James M. , Gralow Julie R. , Levy Ronald , Miller Richard A. 
Published: August 2005

AbstractPeripheral blood mononuclear cells from ten normal donors were labeled with a monoclonal antibody specific for monocytes and analyzed using a fluorescence activated cell sorter (FACS). Forward and 90° light scatter parameters were studied in order to apply optimal computerized gating to identify and exclude monocytes from lymphocyte populations. An average of 9.45% versus 1.22% of cells, within chosen lymphocyte gates established by forward angle and 90° scatter, respectively, were identified as monocytes. In samples from ten donors, the exclusion of monocytes from the lymphocyte population was more efficient using 90° scatter than forward scatter. Simultaneous use of forward and 90° scatter did not significantly improve the ability to accurately exclude monocytes, but did result in a significant increase in the improper exclusion of lymphocytes. Use of 90° scatter alone, forward scatter alone, and forward and 90° scatter simultaneously to identify lymphoid cells resulted in the exclusion of 12, 17, and 23% of lymphocytes from further analysis. The 90° scatter alone appears to be the optimal method to eliminate monocytes electronically from mononuclear cell populations in which lymphocytes are being studied.
Detection of distinct subpopulations of langerhans cells by flow cytometry and sorting
Authors: Vaigot P. , Czernielewski J. , Prunieras M. 
Published: August 2005

AbstractFlow cytometry was found to be a very appropriate tool for the study of Langerhans cells (LC), which represent a minor cell population (2–3%) of human epidermis, and allowed us to obtain new phenotypic, functional and cell cycle data on these rare cells.The phenotypic analysis of cell surface antigens demonstrates the existence of two subpopulations of LC: the former is HLA‐DR+ and OKT 6 + (about 90% of total HLA‐DR+ cells) and the latter is HLA‐DR+ and OKT 6− (about 10% of total HLA‐DR+ cells). These subpopulations of LC are both able to stimulate the proliferation of peripheral blood lymphocytes (PBL) in the presence of keratinocytes i.e., in mixed skin lymphocyte reaction (MSLR).Analysis of the cell cycle could be performed on OKT 6+ LC. Results show that they can be found in the various phases of the cell cycle, suggesting that the large majority of Langerhans cells are able to proliferate in situ in normal human epidermis.
DNA image cytometry on machine‐selected breast cancer cells and a comparison between flow cytometry and scanning cytophotometry
Authors: Cornelisse Cornelis J. , Van Driel‐Kulker Anneke M. 
Published: August 2005

AbstractA DNA image cytometry method, implemented on the LEYTAS image processing system, has been applied to acriflavine‐Feulgenstained breast cancer cytology specimens. An essential feature of the LEYTAS image cytometry method (LCM) is the automated selection of single nuclei according to predetermined specifications. Visual interaction has been used to reject remaining artefacts like overlapping nuclei. DNA profiles obtained with LCM have been compared with DNA profiles obtained by scanning cytophotometry (SCM) or flow cytometry (FCM). The resolution of DNA profiles obtained with LCM is similar to that from SCM but lower than that from FCM. However, a high correlation is found for the DNA indices measured with LCM and FCM (r = 0.97). The LCM profiles of aneuploid tumours generally showed lower accessory diploid fractions than FCM profiles due to the automated rejection of leukocyte nuclei. Also, LCM profiles frequently showed the presence of minor subpopulations of highly aneuploid/polyploid tumour cells that could not be identified by FCM. Therefore, LCM appears to be supplementary to FCM for studying tumour cell stemline heterogeneity.
Spot‐blot analysis of sorted chromosomes assigns a fructose intolerance disease locus to chromosome 9
Authors: Lebo Roger V. , Tolan Dean R. , Bruce Barry D. , Cheung Mei‐Chi , Kan Yuet W. 
Published: August 2005

AbstractThe aldolase B gene was mapped to chromosome 9 using a rapid gene mapping system. This system uses a dual‐laser sorter to identify and separate metaphase human chromosomes stained with either DIPI‐chromomycin or Hoechst‐chromomycin. Chromosome panels were constructed from a normal cell line by sorting 22 chromosome fractions directly onto nitrocellulose filters. Twelve labeled gene probes hybridized to the sorted chromosomal DNA fractions predicted by previous chromosome assignments. Eighteen newly cloned genes have been mapped using the same protocol.
The relationship between mean channel selection and the calculated coefficient of variation
Authors: Schuette W. H. , Carducci E. , Marti G. E. , Shackney S. E. , Eden M. 
Published: August 2005

AbstractCalculated coefficients of variation (CV) taken from the quotient of the standard deviation (S.D.) and the mean value of measured distributions are often used as an indicator of system performance in linear flow cytometry (FCM). The ability of the calculated CV to estimate the true CV of the underlying experiment before grouping (channelization) is dependent on the relationship between the width of the data channels and the magnitude of the S.D. of the measured distribution. When the channel width is equal to the S.D. of a distribution, the calculated CV is approximately 20% larger than the true CV of an experiment. By the time the S.D. is only one‐half of a channel width, the calculated CV is unreliable. When the distribution S.D. is narrower than a channel's width, small changes in the distribution mean value will cause large variations in the calculated CV. As the true CV decreases, the calculation must be made with higher mean channel values. This dependence of calculated CV accuracy upon the relationship between S.D. and channel width places limitations upon mean channel selection that must be considered when using CV calculations for evaluating system performance, especially when looking for small improvements during optical alignment procedures. When an instrument is assumed to have a constant CV and the data are collected linearly, it is possible to improve the CV estimation accuracy by placing distributions in higher‐numbered channels. When linear data with a constant CV are processed logarithmically before channelization, the accuracy of linear equivalent CV calculations is dependent upon the slope of the logarithmic converter and the number of channels available. Under these conditions, CV determination accuracy is independent of mean channel selection.
A fast cell sampler for flow cytometry
Authors: Battye F. L. , Darling W. , Beall J. 
Published: August 2005

AbstractA simple device has been developed for delivering samples into a flow cytometer. Designed with economy, simplicity, and flexibility in mind, this device, having only one moving part, can be used for sample volumes as small as 20 μl, for virtually any form of cell sample container, and for a wide range of cell concentrations. It consists essentially of a lever‐operated disc valve that allows the cell sample to be loaded into a loop of tubing and then to be injected into the cytometer nozzle under pressure from a saline source. The sampler has lifted the maximum analytical throughput of a FACS II cell sorter to better than 120 samples per hour.
Effect of 5‐fluoro‐2′‐deoxyuridine (FdUrd) on 5‐bromo‐2′‐deoxyuridine (BrdUrd) incorporation into DNA, measured with a monoclonal BrdUrd antibody and by the BrdUrd/hoechst quenching effect
Authors: Ellwart Joachim , Dörmer Peter 
Published: August 2005

AbstractThe purpose of this study was to improve the application of bromodeoxyuridine (BrdUrd) for the flow cytometric analysis of cell kinetics. In order to obtain a quantitative measure of the DNA synthesis rate (or the number of divided cells). BrdUrd should replace thymidine (dThd) completely in the newly synthesized DNA strands. The de novo synthesis of dThd monophosphate competing with BrdUrd incorporation was stopped by fluorodeoxyuridine (FdUrd). Cells of a human leukemic cell line (REH) were exposed O BrdUrd for either 20 min, 8h, or 24h. Bromodeoxyuridine incorporation was determined by a monoclonal antibody as well as by the BrdUrd/Hoechst (H) technique. Counter‐staining of the DNA was performed with propidium iodide or ethidium bromide. DNA fluorescences were measured in both techniques with a two‐parameter flow cytometer, the his‐tograms being analyzed by computer. It was found that FdUrd is required in the BrdUrd/H technique for replacement of dThd at low BrdUrd concentrations and long incubation times. With short incubation periods, as used for detection by the monoclonal anti‐BrdUrd antibody, FdUrd increases the incorporated BrdUrd amount when BrdUrd concentrations of 10 μU or less are applied.