bluuebunny/crossref_metadata_2025_split huggingface dataset:
Flow cytometric evaluation of DNA stainability with propidium iodide after histone H1 extraction
Authors: Giangarè Maria Chiara , Prosperi Ennio , Pedrali‐Noy Guido , Bottiroli Giovanni 
Published: August 2005

AbstractA flow cytometric evaluation of the effect of the histone H1 extraction on DNA stainability with propidium iodide was performed on isolated HeLa nuclei. Selective removal of the lysine‐rich protein was attained by using two established techniques involving treatment with 0.7 M NaCl or low pH. DNA stainability was monitored at different dye/DNA‐P ratios, varying from low to high saturating concentrations. Depletion of the histone H1from nuclei results in the transition from low to high affinity of a portion of binding sites, as shown by (1) the increase in fluorescence intensity after staining with the dye at low saturating concentrations and (2) the higher value of the fluorescence intensity ratio (FI5FI50) exhib ited by H1‐depleted nuclei stained with a low (5 μg/ml) vs. a high (50 μg/ml) concentration, as compared with control samples.
Simultaneous flow cytometric analysis of surface markers and nuclear Ki‐67 antigen in leukemia and lymphoma
Authors: Drach Johannes , Gattringer Claus , Glassl Herta , Schwarting Roland , Stein Harald , Huber Heinz 
Published: August 2005

AbstractThe monoclonal antibody Ki‐67 identifies an antigen present during the late G1, S, G2, and M phases of the cell cycle, whereas resting cells do not express this antigen. Immunostaining with Ki‐67 provides a simple method with which to determine the growth fraction of a malignant cell population without requiring a laborious procedure or use of radioactive materials. Thus far, detection of Ki‐67–positive cells by flow cytometry was limited because of nuclear location of the antigen. In this study, periodate‐lysine‐paraformaldhyde (PLP) fixation of cells in suspension, labeling with Ki‐67, and the subsequent flow cytometric analysis of the tumor growth fraction is described. Fixation with PLP at −10°C for 15 min rendered the plasma membrane permeable without destroying cell surface antigens. Thus double immunofluorescence studies using both a surface marker and Ki‐67 could be performed. This offers the additional advantage of being able to define the phenotype of proliferating cells. This method was applied to determine the growth fraction in peripheral blood and bone marrow samples of patients with leukemia and non‐Hodgkin's lymphoma. The results of Ki‐67 studies in 91 patients are shown. A wide variability of individual Ki‐67 values was observed within each entity. Use of this flow cytometric procedure substantially facilitates the quantification of proliferating cells in pathological blood and bone marrow samples.
Use of xantham gum to suspend large particles during flow cytometric analysis and sorting
Authors: Freyer James P. , Fillak David , Jett James H. 
Published: August 2005

AbstractIn this report we describe the use of xantham gum as a biologically inert material for increasing the viscosity of a suspension of cells or particles during flow cytometric analysis and sorting. A 0.1% concentration of xantham gum in culture medium or saline will increase the viscosity ∼︁9‐fold. For suspensions of multicellular spheroids 100–400 μm in diameter the measured sedimentation velocity was ∼︁9 times slower than that in medium alone. Thus, spheroids of 100 μm diameter remain in suspension in 0.1% xantham gum for 66 min, compared to 7.5 min in culture medium. This allows extended periods of sorting without stirring or agitating the sample suspension. The xantham gum solution is noncytotoxic for periods up to 8 h as measured by clonogenicity assay. Xantham gum has the added advantage that the viscosity is significantly reduced when the solution is subjected to shear stress, such as during flow. This technique should be applicable to extended sorting of suspensions of spheroids, plant cells, and other large particles, as well as for analyzing and sorting single cells for extended periods.
Modified box‐cox transform for modulating the dynamic range of flow cytometry data
Authors: Dvorak James A. , Banks Steven M. 
Published: August 2005

AbstractWe describe an algorithm,

based upon Box‐Cox transformations as an alternative to nonlinear electronic amplifiers to expand or compress high‐ or low‐amplitude flow cytometer‐derived signals. If the indexing parameter λ < 1, input channels in the high‐amplitude input range are compressed in the output range as occurs when an electronic logarithmic amplifier is used. However, if λ > 1, input channels in the low‐amplitude input range are compressed in the output range as occurs when an electronic power amplifier is used. Our modified Box‐Cox transform can be implemented either during data collection or off‐line for the transformation of previously collected raw data. The transform is the equivalent of an infinite class of nonlinear amplifiers. As the transform is implemented in software, it does not suffer from many of the disadvantages of nonlinear electronic amplifiers.
A unified model of eukaryotic chromosomes
Authors: Manuelidis Laura , Chen Terence L. 
Published: August 2005

AbstractA revised model of DNA packaging into chromosomes is presented. Its features are consistent with observed structural dimensions and the molecular periodicities related to transcription, replication and matrix attachment domains. Thetransitions between euchromatic, heterochromatic and metaphase states are explained simply. Molecular and physical properties of chromosomal bands, and their correlation with specific DNA sequence motifs are discussed.
Direct nonradioactive in situ hybridization of somatic cell hybrid DNA to human lymphocyte chromosomes
Authors: Kievits T. , Devilee P. , Wiegant J. , Wapenaar M. C. , Cornelisse C. J. , van Ommen G. J. B. , Pearson P. L. 
Published: August 2005

AbstractBiotinylated DNA from various human–rodent hybrids was hybridized to human lymphocyte spreads after preannealing of the repeated sequences with sonicated total human DNA. Fluorescent labeling was achieved by successive treatments with fluorescein‐labeled avidin and biotinylated antiavidin antibody. The use of labeled total DNA from hybrids with known chromosome composition permits the fluorescent staining‐(“painting”) of specific chromosomes, or parts thereof, in human lymphocyte metaphases. Alternatively, the human chromosome content of cell hybrids with unknown chromosome composition is directly assessed from the labeling pattern of human lymphocyte spreads using the total hybrid DNA as probe.
High resolution mapping of Xenopus laevis 5S and ribosomal RNA genes by EM in situ hybridization
Authors: Narayanswami Sandya , Hamkalo Barbara A. 
Published: August 2005

AbstractWe have developed a modification of in situ hybridization at the electron microscope level that permits simultaneous detection of at least two sequences. Probes are labelled with either biotin or AAF and detected with two distinct sizes of colloidal gold. This protocol has been applied to map the positions of Xenopus laevis oocyte‐type 5S genes relative to ribosomal precursor genes in several independently derived cell lines. The results for the line TRXO, which expresses some oocyte 5S RNA, indicate that this inappropriate expression is not due to translocation from telomeric sites into the nucleolus organizer, as previously hypothesized (7). In addition we found that four other Xenopus cell lines, none of which express these genes, also contain distinct, 5S oocyte translocations. These results suggest that an alteration in chromosome position is insufficient to result in gene activation and that sequences which are telomeric‐proximal are exceptionally prone to translocation.
Flow cytometric quantification of human chromosome specific repetitive DNA sequences by single and bicolor fluorescent in situ hybridization to lymphocyte interphase nuclei
Authors: van Dekken H. , Arkesteijn G. J. A. , Visser J. W. M. , Bauman J. G. J. 
Published: August 2005

AbstractFluorescent in situ hybridization allows for rapid and precise detection of specific nucleic acid sequences in interphase and metaphase cells. We applied fluorescent in situ hybridization to human lymphocyte interphase nuclei in suspension to determine differences in amounts of chromosome specific target sequences amongst individuals by dual beam flow cytometry. Biotinylated chromosome 1 and Y specific repetitive satellite DNA probes were used to measure chromosome 1 and Y polymorphism amongst eight healthy volunteers. The Y probe fluorescence was found to vary considerably in male volunteers (mean fluorescence 169, S.D. 35.6). It was also detectable in female volunteers (mean fluorescence 81, S.D. 10.7), because 5–10% of this repetitive sequence is located on autosomes. The Y probe fluorescence in males was correlated with the position of the Y chromosome cluster in bivariate flow karyotypes. When chromosome 1 polymorphism was studied, one person out of the group of eight appeared to be highly polymorphic, with a probe fluorescence 26% below the average. By means of fluorescent in situ hybridization on a glass slide and bivariate flow karyotyping, this 26% difference was found to be caused by a reduction of the centromere associated satellite DNA on one of the homologues of chromosome 1.The simultaneous hybridization to human lymphocyte interphase nuclei of biotinylated chromosome 1 specific repetitive DNA plus AAF‐modified chromosome Y specific DNA was detected by triple beam flow cytometry. The bicolor double hybridized nuclei could be easily distinguished from the controls. When the sensitivity of this bicolor hybridization is improved, this approach could be useful for automatic detection of numerical chromosome aberrations, using one of the two probes as an internal control.
A computer program for analyzing bivariate flow karyotypes
Authors: van den Engh Ger , Hanson Don , Trask Barb 
Published: August 2005

AbstractThis article describes a computer program for analyzing bivariate flow karyotypes of human chromosomes stained with Hoechst 33258 (HO) and chromomycin A3 (CA). The karyotype first is divided into regions that contain chromosome peaks. The chromosomes that are associated with those areas are identified. The distributions in these areas then are fitted with mathematical functions of increasing complexity. The process starts by fitting a specified number of univariate Gauss functions to projections of the HO and CA distributions of each area. The final fit can include multiple bivariate Gauss functions, including a background function for debris subtraction. The results of one stage in the fitting process serve as seed values for the next, more complex step. Since the program autonomously estimates the starting values for the iterative fitting procedures, the fit results are insensitive to operator bias and the program will consistently converge to the same solutions. The resulting table of parameter values can be used to compare flow karyotypes to a reference data set.
Clinical applications of flow karyotyping in myelocytic leukemia by stimulation of different subpopulations of cells in blood or bone marrow samples
Authors: Arkesteijn G. J. A. , van Dekken H. , Martens A. C. M. , Hagenbeek A. 
Published: August 2005

AbstractExamples are presented in which normal as well as abnormal chromosome distributions could be obtained from the same individual by means of bivariate flow karyotyping. Selective stimulation of T‐lymphocytes obtained by E‐rosetting from the blood of a patient with acute myelocytic leukemia resulted in a normal flow karyogram. The specific stimulation of myelocytic leukemia cells with granulocyte‐macrophage colony stimulating factor (GM‐CSF) and interleukin 3 (IL‐3) yielded flow karyograms displaying the leukemia‐associated chromosome abnormalities. The resulting flow karyograms could be used to discriminate between homolog differences, which appear normally in virtually every individual, and leukemia‐associated chromosomal aberrations. In the case of a female chronic myelocytic leukemia patient who received bone marrow form an HLA‐identical male donor, specific stimulation of various subsets of cells enabled to discriminate between leukemic host cells and non‐leukemic donor cells. Both the leukemia‐specific translocations and sex chromosomes were used as markers to analyse the flow karyograms obtained from the same sample.
High gradient magnetic cell separation with MACS
Authors: Miltenyi Stefan , Müller Werner , Weichel Walter , Radbruch Andreas 
Published: August 2005

AbstractA flexible, fast and simple magnetic cell sorting system for separation of large numbers of cells according to specific cell surface markers was developed and tested. Cells stained sequentially with biotinylated antibodies, fluorochrome‐conjugated avidin, and superparamagnetic biotinylated‐microparticles (about 100 nm diameter) are separated on high gradient magnetic (HGM) columns. Unlabelled cells pass through the column, while labelled cells are retained. The retained cells can be easily eluted. More than 109 cells can be processed in about 15 min. Enrichment rates of more than 100‐fold and depletion rates of several 1,000‐fold can be achieved. The simultaneous tagging of cells with fluorochromes and very small, invisible magnetic beads makes this system an ideal complement to flow cytometry. Light scatter and fluorescent parameters of the cells are not changed by the bound particles. Magnetically separated cells can be analysed by fluorescence microscopy or flow cytometry or sorted by fluorescence‐activated cell sorting without further treatment. Magnetic tagging and separation does not affect cell viability and proliferation.
Multiparametric analysis of cell membrane permeability by two colour flow cytometry with complementary fluorescent probes
Authors: Dive Caroline , Watson James V. , Workman Paul 
Published: August 2005

AbstractWe describe an improved twin‐probe multiparameter flow cytometric technique to examine cell membrane permeability. Ability to retain preloaded intracellular bis‐carboxyethyl carboxy fluorescein (BCECF, green fluorescence) and to exclude extracellular propidium (red fluorescence) is measured, simultaneously with forward and right‐angle scatter. This has significant advantages over an earlier method using fluorescein together with ethidium. In addition to the two expected cell populations which were stained green positive, red negative (by convention membrane “intact” and “viable,” Region 1) and green negative, red positive (“membrane‐damaged” and “non‐viable,” Region 3), a third population was seen which fluoresced neither green nor red and displayed intermediate light scatter characteristics (Region 2). This was true for each of 9 cell types in vitro. For EMT6 mouse mammary tumour cells held under sub‐optimal conditions or treated with membrane‐active drugs, progression from Region 1 to Region 2 was observed, followed by further progression from Region 2 to Region 3. Cells eventually accumulated in Region 3. These results suggest that sequential changes in membrane structure lead to increased permeability, first with respect to intracellular BCECF and in turn to extracellular propidium.
Characteristic immunophenotyping artefact seen in patients with anti‐mouse immunoglobulin antibodies
Authors: Wilks D. , Byrom N. , Walker L. , Habeshaw J. , Dalgleish A. 
Published: August 2005

AbstractPatients treated with murine monoclonal antibodies develop anti‐mouse immunoglobulin antibodies which can interfere with flow cytometry of lymphocytes, giving rise to high levels of non‐specific staining. This artefact can be avoided by using separated, washed lymphocytes for immunophenotyping.
Chlorophyll α fluorescence measurements of isolated spinach thylakoids obtained by using single‐laser‐based flow cytometry
Authors: Xu Chunhe , Auger Julie , Govindjee  
Published: August 2005

AbstractFlow cytometry data of spinach thylakoid membrane preparations indicate the presence of a homogeneous thylakoid population. Fluorescence data from a flow cytometer and comparison with data from two other fluorometers show that cholorophyll α fluorescence detected with a flow cytometer has the character of maximum fluorescence (Fmax), not of the constant component (Fo). This conclusion is important since Fo measures fluorescence that is affected mostly by changes in excitation energy transfer and Fmax‐Fo (the variable fluorescence) by changes in photochemistry. This was demonstrated by: (1) The light intensity as well as diffusion rate dependence of the quenching effect of various quinones (p‐benzoquinone, phenyl‐benzoquinone, and 2,5‐dibromo‐3‐methyl‐6‐isopropyl‐p‐benzoquinone, DBMIB) on fluorescence yield; quenching for the same concentration of these quinones was lower at the higher than at the lower light intensities. (2) Temperature dependence of the fluorescence yield; increasing the temperature from 20 to 70°C did not show an increase in fluorescence yield using a flow cytometer in contrast to measurements with weak excitation light, but similar to those obtained for Fmax. (3) Addition of an inhibitor diuron up to 100 μM did not change the fluorescence intensity. A comparison of quenching of fluorescence by various quinones obtained by flow cytometry with those by other fluorometers suggests that the high intensity used in the cytometry produces unique results: the rate of reduction of quinones in much larger than the rate of equilibration with the bulk quinones.
Flow cytometric recognition of clastogen induced chromatin damage in G0/G1 lymphocytes by non‐stoichiometric Hoechst fluorochrome binding
Authors: Kubbies M. 
Published: August 2005

AbstractChanges in chromatin structure were induced in human peripheral blood lymphocytes. Resting G0/G1 cells were exposed to either X‐rays, mitomycin C, or bleomycin and stimulated with PHA. Exposure to such agents provokes an increase in the non‐cycling cell fraction; and a distinctive, non‐cycling G‐/G1 subpopulation appears which is characterized by a 23% reduced Hoechst fluorescence intensity. This novel subpopulation was found as early as 24 h after PHA stimulation; it was still present in 72 h cultures. Bromodeoxyuridine (BrdUrd/Hoechst) 33258‐ethidium bromide (EB) flow cytometric analysis revealed increments of this subpopulation from 2% of the non‐cycling cell fraction in the control culture to 29% (X‐rays), 15% (mitomycin C), and 24% (bleomycin) after clastogen exposure. In the presence of the ligase inhibitor 3‐aminobenzamide, this aberrant cell population increased significantly after X‐ray treatment. With the aid of a viable BrdUrd/Hoechst staining assay, the newly identified non‐cycling subpopulation with decreased Hoechst 33258 binding was identified as a distinctive signal cluster. Other than the regular non‐cycling and cycling cell fractions, this subpopulation with non‐stoichiometric Hoechst dye binding showed progressive uptake of ethidium bromide; however, by such criteria 44% of the subpopulation was still viable. It is concluded that the clastogen induced subpopulation of noncycling cells represents damaged cells with altered dye binding properties.
Bromodeoxyuridine incorporation into DNA of human leukemia cells is not concentration dependent
Authors: Powell Bayard L. , Gregory Betsy W. , Kute Timothy E. , Morgan Timothy M. , Lyerly E. Susan , Capizzi Robert L. 
Published: August 2005

AbstractLeukemia blasts isolated from bone marrow aspirates of 44 adults with acute leukemia were incubated for 1 h with 0.008–32 μM bromodeoxyuridine (Brd‐Urd). After dual labeling with monoclonal anti‐BrdUrd antibodies and propidium iodide, the cells were analyzed by flow cytometry. Percent labeled cells and intensity of labeling were similar over concentrations of BrdUrd ranging from 0.8–32 μM—a 40‐fold range. Therefore, despite potential interpatient variability in nucleoside pharmacokinetics, commonly used doses of BrdUrd which are intended to achieve steady‐state plasma concentrations in the 8.0 μM range can be expected to provide a reliable estimate of the S‐phase fraction.
Detection of S cells and evaluation of DNA denaturation protocols by image cytometry of fluorescent BrdUrd labelling
Authors: Humbert Catherine , Giroud Françoise , Brugal Gérard 
Published: August 2005

AbstractThis paper addresses the problem of detecting weak incorporation of BrdUrd and the related efficiency of the denaturation protocols used to unmask this thymidine analog. Evidence is presented that measuring the distribution of BrdUrd‐tagged fluorescence intensities by image cytometry generates a standard deviation threshold that discriminates between positive and negative MRC5 cells in vitro. A comparison of the thresholding by standard deviation (SDT) with the usual thresholding by the nuclear total fluorescence intensity (FIT) demonstrated that SDT has a significantly higher sensitivity (99.4–100%, depending on the denaturation protocols) than FIT (94.7 and 74.3%, respectively), although both tests have a high specificity (93% and 100%, respectively) for detecting S cells. Since detecting the S cells is not only dependent on the test used, but also on the denaturation protocols, a quality index (QI) was derived from the standard deviation and the mean value of the nonspecific fluorescence of negative cell population versus BrdUrd fluorescence of positive cell population. The following DNA denaturation protocols have been assessed according to QI: acidic denaturation, thermal denaturation in formamide, and thermal denaturation in distilled water. Each denaturation procedure was preceded or not by incubation in either proteinase K or Triton X‐100. The results showed that thermal denaturation in formamide, especially when preceded by proteinase K incubation, revealed the largest difference between negative and positive cells. This work also demonstrated that image cytometry of BrdUrd‐labelled cells can be suitable for clinical application because of the high sensitivity provided and the small samples needed.
Evaluation of immunocytochemical detection methods of incorporated bromodeoxyuridine in hybridomas
Authors: Williams Lance S. , Nordberg Judith A. , Chau Pao C. 
Published: August 2005

AbstractBivariate distributions obtained fron nominal acid hydrolysis or thermal treatment methods used in the cell cycle analysis of incorporated bromodeoxyuridine were shown to be unacceptable with hybridomas. Four different cell treatment and staining methods were compared. These methods are acid hydrolysis, thermal denaturation, nuclei extraction with pepsin digestion, and simultaneous pepsin digestion and acid hydrolysis. The nuclei extraction method was determined to be the most appropriate for the immunocytochemical staining of incorporated bromodeoxyuridine in hybridomas. The resulting bivariate distribution provides a clear distinction between labelled and unlabelled cell fractions. The method based on nuclei extraction with pepsin digestion was optimized for a hybridoma line used in this study.
Simultaneous flow cytometric detection of cellular c‐myc protein, incorporated bromodeoxyuridine, and DNA
Authors: Rosette Caridad D. , DeTeresa Patricia S. , Pallavicini Maria G. 
Published: August 2005

AbstractWe describe a multivariate flow cytometric technique for simultaneous analysis of specific nuclear protein, bromodeoxyuridine (BrdUrd) incorporated into DNA and DNA content in single cells in suspension. The procedure involves fixation of BrdUrd‐exposed cells with paraformaldehyde, heat denaturation of cellular DNA, followed by sequential immunochemical reactions to label incorporated BrdUrd and nuclear protein, and finally staining of total DNA with propidium iodide. The cells are analyzed flow cytometrically and multivariate data acquired in list mode to facilitate analyses of heterogeneous subpopulations. We applied this technique to measure c‐myc protein, incorporated BrdUrd, and DNA content in subpopulations present in a recombinant Chinese hamster ovary (CHO) cell line carrying approximately 800 copies of murine c‐myc sequences under control of an inducible heat shock promoter.