bluuebunny/crossref_metadata_2025_split huggingface dataset:
Experimental parameters and a biological standard for acridine orange detection of drug‐induced alterations in chromatin condensation
Authors: Block Adrienne L. , Bauer Kenneth D. , Williams Thomas J. , Seidenfeld Jerome 
Published: August 2005

AbstractWe investigated a number of sample‐preparative parameters for use of flow cytometry to detect chromatin condensation in cells stained with acridine orange after DNA in situ is partially denatured by acid treatment. Stability and data reproducibility for both control and drug‐treated ME‐180 and HT‐29 cells were assessed over: (1) a range of cell concentrations in 2.56 × 10−5 M acridine orange; (2) 15 days of storage in fixative; (3) various times between RNase digestion and staining; and (4) increasing times between staining and analysis. Listmode data for red and green fluorescence were collected and mean fluorescence intensities of G1, S, and G2 subpopulations of HT‐29 and ME‐180 cells were computed. These were normalized to data from HeLa‐S3 cells and fluorescent microspheres to control for inter‐experiment variations in staining and instrumental parameters, respectively. The normalized red and green fluorescence data were used to calculate αt for G1 cells [αt = red fluorescence/(total fluorescence)]. Exponentially growing HeLa‐S3 cells were a very consistent and reproducible biological standard to control for fixation and staining variability. Mean fluorescence intensities of control and difluoromethylornithine‐treated (i.e., polyamine depleted) cells remained stable and reproducible across all tested ranges for cell concentration, storage in fixative, and time after RNase digestion. This technique can thus be used to evaluate difluoromethylornithine‐induced changes in chromatin condensation of samples stored for as long as 2 weeks and analyzed all on 1 day.
Simultaneous increased expression of E‐rosette receptor (CD2, T11) and T cell growth factor receptor on human T lymphocytes during activation
Authors: Redelman Doug 
Published: August 2005

AbstractThe E‐rosette receptor (CD2, T11) is a differentiation antigen expressed on immature and mature human T lymphocytes. Activation of T cells from human peripheral blood with phytohemagglutinin (PHA) or with monoclonal antibody to the CD3‐Ti complex (anti‐Leu‐4) caused the expression of CD2 to increase 10‐ to 20‐fold. Dual parameter correlated analyses with antibody to the T cell growth factor (TCGF) receptor (anti‐Tac) and anti‐CD2 antibody demonstrated that the increase in CD2 expression occurred at the same time and on the same cells that expressed the TCGF receptor after stimulation with PHA. The increased expression of CD2 and the initial expression of Tac were totally inhibited by cycloheximide, but were not affected by sufficient actinomycin‐D to block the T cell proliferative response. The expression of CD2 was compared with the expression of CD4 and CD8, i.e., T cell differentiation antigens on cytotoxic/suppressor or helper T cell, respectively. Althouth virtually all of the small percentage of freshly isolated Tac+ peripheral blood cells belonged to the CD4+, CD8− subset, both CD4+ and CD8+ T cells were equivalently activated by PHA to express Tac. By 20–30 hr after activation, the expression of CD4 or CD8 was initially decreased 10–50%. Subsequently, the expression of CD4 and CD8 returned to the levels on resting T cells but did not increase further. Therfore, the increase in CD2 expression does not reflect a universal property of cell surface antigens on activated T lymphocytes.
On multiparameter data analysis in flow cytometry
Authors: Mann Reinhold C. 
Published: August 2005

AbstractIncreasing numbers of parameters that are accessible to simultaneous measurement in flow cytometric instruments, combined with the extremely large sample sizes common in flow cytometry, make it necessary to examine methods of multivariate statistics for their applicability to problems of visualization and quantitative analysis of flow cytometric data. This article describes some approaches to dimensionality reduction that appear well suited for data sets obtained by flow cytometry.
Direct hybridization to DNA from small numbers of flow‐sorted nucleated newborn cells
Authors: Bianchi Diana W. , Harris Peter , Flint Alan , Latt Samuel A. 
Published: August 2005

AbstractA technique is described that allows direct hybridization to the DNA of cells flow sorted onto nitrocellulose filters, which obviates an intervening DNA isolation step. The feasibility of this technique for studying small numbers of cells is demonstrated with human cord blood, which has a high proportion of nucleated cells. The cells are stained with fluorescein‐conjugated anti‐HLe‐l, a monoclonal antibody that recognizes mature leucocytes. Anti‐HLe‐l‐positive cells are all nucleated, and a controlled, precise number of them may be sorted directly onto a nitrocellulose membrane. In cord blood, a small percentage of anti‐HLe‐l‐negative cells are nucleated erythrocytes, which may also serve as a source of DNA.Studies were performed on male or female newborn cells flow sorted onto introcellulose membranes and hybridized with either a nonspecific human repeat DNA probe or a Y chromosome‐specific probe. Importantly, the sex of the newborn could be determined at the DNA level from as few as 50 sorted cord blood leucocytes or 5,000 HLe‐l‐negative cells. Since nucleated erythrocytes are common in fetal blood but rarely found in the peripheral circulation of adults, the method has potential application for the determination of fetal sex from analysis of flow‐sorted nucleated erythrocytes present in the maternal circulation during pregnancy.
Cytometry of breast carcinoma: Significance of ploïdy balance and proliferation index
Authors: Opfermann Martina , Brugal Gérard , Vassilakos Pierre 
Published: August 2005

AbstractImage cytometry of DNA distribution in fine needle biopsies of breast carcinomas at first diagnosis was performed to see if there were significant differences in DNA histograms between patients having very different outcome but same tumor histological typing and similar therapy. Two groups of patients were considered retrospectively: the first (20 patients) with survival time shorter than 5 years and the second (20 patients) with survival time longer than 10 years. Seven benign tumors were used as controls. Ten ploïdy classes were defined. The frequencies of cells in those classes were used as independent features in a supervised multivariate analysis. The advantages of this approach was pointed out with respect to the four‐type classification of Auer. The scattering of DNA histograms within the feature space showed that a subgroup of patients with poor prognosis was clearly separated from a subgroup of patients with good prognosis but both long survival patients and short survival patients were scattered in between. In order to replace the multivariate classification of histograms by a simpler approach, two parameters were computed which explained most of the scattering in the feature space: the ploïdy balance (difference between the percentages of euploid and aneuploïd cells) and the proliferation index (percentage of cells between peaks). The scattergram of patients according to these parameters showed again that some DNA distributions were specific for either good or bad prognosis. But the separation was uncertain for seven short‐survival patients and six long‐survival patients. For six patients, the DNA distributions were very similar between long and short survival times. Those patients thus could not be separated even by means of discriminant analysis. The main conclusion of this study was that, for a significant number of patients, the objective multivariate classification of tumors DNA profiles is of little assistance to the pathologist who has to give a prognosis for the one patient under consideration.
DNA ploidy and survival in breast cancer patients
Authors: Cornelisse C. J. , van de Velde C. J. H. , Caspers R. J. C. , Moolenaar A. J. , Hermans J. 
Published: August 2005

AbstractFlow cytometric DNA ploidy measurements using frozen or deparaffinized tumor specimens were performed on 565 primary breast cancers from patients treated in the period 1975–1984. Twenty‐nine percent of the cases were diploid, 61% had a single aneuploid stemline, and 10% were multiploid. Aneuploid tumors more often had negative estrogen receptor values than diploid tumors, but no significant correlation was found between ploidy class and TNM stage. Patients with more than ten positive axillary lymph nodes had predominantly aneuploid tumors. Overall and distant relapse‐free survival were higher for patients with diploid tumors and low‐aneuploid tumors. Stratification of the patients according to degree of lymph node involvement, TNM stage, and menopausal stage showed that the prognostic effect of aneuploidy was apparent predominantly in patients with locally advanced disease. Postmenopausal node‐positive patients with diploid tumors had a significantly better prognosis than those with aneuploid tumors, but this difference was not found for the comparable premenopausal group. Multivariate analysis with the Cox proportional hazards model indicated that ploidy is an additional, independent prognostic factor in postmenopausal patients.
Immunochemical analysis of the structure and function of chromosomal proteins
Authors: Bustin Michael 
Published: August 2005

AbstractImmunochemical appxroaches are useful in studying the nuclear organization and cellular function of chromosomal components. Antibodies specific to histones and to defined nonhistone proteins have been used to study nucleosome heterogeneity, to visualize the presence of histone in transcriptionally active chromatin, and to isolate DNA sequences associated with specific chromosomal proteins.
Purification and analysis of rat hematopoietic stem cells by flow cytometry
Authors: McCarthy Kenneth F. , Hale Martha L. , Fehnel Paula L. 
Published: August 2005

AbstractThe monoclonal antibody OX7 recognizes an epitope expressed on the Thy‐1 glycoprotein, OX22 recognizes the high molecular weight forms(s) on leukocyte common antigen, and W3/13 recognized determinants found on certain sialoglycoproteins. Recently, the rat colony‐forming unit spleen (CFU‐S) was characterized as being OX7 upper 20% positive (OX7u20%), OX22 negative (OX22−), and W3/13 weakly positive (W3/13+). In the present study these observations have been extended to include the hematopoietic stem cell (HSC). Rat marow cells were incubated with allophycocyanine‐OX7 Fab' (APC‐OX7 Fab') and phycoerythrin (B‐OX22) Fab' (PhyB‐OX22 Fab'). The cells were sorted with a FACS‐ii instrument by using a Krypton laser tuned to the 530 nm spectral line for phycobiliprotein excitation. It was found that marrow cells capable of protecting lethally irradiated Lewis rats (9.5 Gy total body radiation, 0.4 Gy/min Co60) had the phenotype OX7u20%, OX22−. The percentage of cells in the marrow with this phenotype was found to be 0.34 ± 0.01 (mean ± S.E.). Three thousand of these cells were required to rescue 50% of lethally irradiated recipients (30‐d survival), while the number of unsorted bone marrow cells required was 1.05 × 106. Thus, a 350‐fold purification of the HSC was realized. Although CFU‐S copurified with HSC, purification of only 105‐fold was obtained. This might indicate that purified HSC have a reduced capacity to generate splenic hematopoietic colonies. The OX7u20%, OX22−‐enriched HSC population could be further divided into W3/13 dimand W3/13+ subpopulations by three‐parameter immunofluorescence analysis with the use of a new optical bench arrangement.
Quantitation of the binding, uptake, and degradation of fluoresceinylated neoglycoproteins by flow cytometry
Authors: Midoux Patrick , Roche Annie‐Claude , Monsigny Michel 
Published: August 2005

AbstractThe fluorescence properties of the fluorescein residues bound to a protein are used to analyze by flow cytometry the neoglycoproteins' endocytosis mediated by membrane lectins of Lewis lung carcinoma cells (3LL cells). The quantum yield of fluorescein bound to a protein is dependent on the number of fluorophore molecules bound to a protein molecule and the pH of the environmental medium. The mean fluorescence intensity of a fluorescein molecule bound to a protein decreases when the number of fluorescein residues per protein molecule increases. However, after proteolytic digestion, the mean fluorescence intensity of a fluorescein molecule is constant and equal to that of free fluorescein. The binding of fluorescein‐labeled α‐glucosylated serum albumin to 3LL cells at 4°C can easily be determined by flow cytometry because under these conditions the environmental pH is neutral, and the neoglycoprotein is not degraded. When the cells are incubated at 37°C in the presence of a fluorescein‐labeled neoglycoprotein, the fluorescence intensity of a cell is low because of the low pH of endosomes and lysosomes but is increased upon a postincubation at 4°C in the presence of monensin, a proton/sodium ionophore. The extent of the proteolytic digestion of an endocytosed neoglycoprotein can be assessed by comparing, upon a monensin postincubation at 4°C, the high cell‐associated fluorescence of cells incubated in the absence of leupeptin (an inhibitor of lysosomal proteases) and the relatively low fluorescence intensity of cells incubated in the presence of leupeptin.
Influence of systematic errors on the evaluation of the S phase portions from DNA distributions of solid tumors as shown for 328 breast carcinomas
Authors: Haag Dietrich , Feichter George , Goerttler Klaus , Kaufmann Manfred 
Published: August 2005

AbstractThe portion of cells in S phase has proved to be a valuable prognostic indicator of early relapse and life expectancy, particularly in breast carcinoma. Comparisons of published data on samples of primary breast carcinoma biopsies showed that the values obtained by analyses of flow cytometric DNA distributions were generally higher than those of determinations based on the tritiated thymidine (3H‐ThdR) labeling index (LI).Flow cytometric DNA analyses of 328 biopsy samples of primary breast carcinomas revealed that these differences could be explained by varying contributions of debris background. Since this influence is inversely proportional to the cell counts in each channel, it may cause considerable errors, particularly in the S phase channels, which normally contain the lowest counts of the DNA distributions. Two different mathematical rationales were tested in order to separate DNA distributions from the debris superimposition. No appreciable differences were found with respect to the essential results. After appropriate subtraction of the background levels, the previously reported discrepancies between cytometrically determined S phase portions and 3H‐ThdR LI values disappeared, and good agreement was achieved for the comparable tumor samples of the present study.In conclusion, debris background subtractions should generally precede the DNA histogram analyses, particularly of solid tumors, in order to obtain reliable S phase values.
Cell cycle perturbation of cultured C6 glioma cells following short‐term contact with a low dose of ACNU
Authors: Genka Shigeru , Shitara Nobuyuki , Tsujita Yoshihiko , Kosugi Yukio , Wu Yaochen , Takakura Kintomo 
Published: August 2005

AbstractThe purpose of this study was to investigate the cell cycle perturbation of cultured C6 rat glioma cells induced by 1‐(4‐amino‐2‐methyl‐5‐pyrimidil) methyl‐3‐ (2‐chlorethyl) 3‐nitrosourea hydrochloride (ACNU) using simultaneous flow cytometric measurements of DNA and bromodeoxyuridine (BrdU) content. A new graphic computer program permitted the quantification of cell density in hexagonal subareas and allowed the fraction of BrdU‐labeled cells with mid‐S phase DNA content (FLS) to be defined in a narrow window. The cell kinetic parameters such as cell cycle time (Tc) and S phase time (Ts) were estimated from a manually plotted FLS curve at 18 and 6 hr, respectively.The major effect of ACNU on the cell cycle was an accumulation of the cells in the G2M phase 12 to 24 hr posttreatment when compared to G2M traverse of untreated cells. For the two‐dimensional analysis, cells were labeled with BrdU and then treated with ACNU, or treated with ACNU and then labeled with BrdU. It was concluded that the cells in the S and G2M phases at the time of ACNU administration progressed to mitosis but that the G1 phase cells accumulated in the subsequent G2M phase. Two‐dimensional FCM analysis using BrdU provided a useful tool in studying cell cycle perturbation.
Flow cytometric evaluation of anti‐herpes drugs
Authors: Rosenthal Ken S. , Hodnichak Cheryl M. , Summers Jack L. 
Published: August 2005

AbstractA rapid means of screening drugs for toxicity and anti‐herpes simplex virus activity was developed based on the flow cytometric detection of HSV induced changes in cellular DNA content. Subconfluent monolayers of human diploid fibroblasts (HEL 299) were assayed for DNA content with propidium iodide 24 h after infection with HSV‐1 (multiplicity of infection 1–10) and treatment with the drug to be tested. Infection was detected by a broadening of the normal diploid and tetraploid peaks and presence of greater than 4‐n DNA staining. Inhibition of viral DNA synthesis and maintenance of the normal growth pattern of control cells was indication of antiviral activity. Toxicity of the compound was indicated by the loss of S phase and tetraploid cell populations. Using this assay, we evaluated the activities of one experimental and two established antiviral agents.
A rapid analytical technique for flow cytometric analysis of cell viability using calcofluor white M2R
Authors: Berglund Deborah L. , Taffs Rolf E. , Robertson Nancy P. 
Published: August 2005

AbstractAnalysis of dead versus live cells is shown to be possible using Calcofluor White M2R (CFW), a fluorescent brightener. Comparison of CFW with both propidium iodide (PI) and fluorescein diacetate (FDA) was performed on a FACS 440 dual laser flow cytometer on several populations of cultured rat and mouse cell lines, peripheral leukocytes, splenocytes, diatoms, and plant protoplasts. As a measure of cell viability, staining results with CFW were strongly associated with PI (correlation coefficient of 0.9886) and FDA (inverse correlation coefficient of 0.9647). With plant and algal cells, controls are necessary as CFW does stain live cells to some extent. CFW (excitation: UV, emission max: 435 nm) can be used in conjunction with two‐color immunofluorescence analysis using fluorochromes excited at 488 nm with no interference.
Solid tumor preparation for flow cytometry using a standard murine model
Authors: Ensley J. F. , Maciorowski Z. , Pietraszkiewicz H. , Klemic G. , Kukuruga M. , Sapareto S. , Corbett T. , Crissman J. 
Published: August 2005

AbstractThe application of flow cytometry (FCM) to solid human tumors has been hindered by the difficulty in producing high yield, viable, unaltered single cell suspensions. Carcinomas containing a high desmosomal content, such as well‐differentiated squamous cell (SCC) cancers of the head and neck (H&N) region, are particularly difficult to prepare. The desire to employ FCM to study cellular DNA parameters of these tumors led to the use of a 3‐methylcholanthrene induced murine SCC for the comparative testing of preparative techniques. Dissociation techniques, including mechanical, enucleation, chemical, single and combination enzymes methods, were comparatively tested. Of these, the combination enzyme treatment employing trypsin and collagenase produced the highest cell yields in the shortest time with the highest dye exclusion viability and the least expense. Several fixation systems including glutaraldehyde, paraformaldehyde, acetic acid, and ethanol were comparatively tested using percent of cell loss and quality of the DNA histograms produced as end points. Ethanol‐water systems with added fetal calf serum provided minimal cell loss and high quality histograms which were stable for extended periods of time. A murine tumor, closely mimicking the histology of the human tumor of interest, may be used as a model for the determination of optimum techniques of solid tumor preparation for flow cytometric analysis.
Solid tumor preparation for clinical application of flow cytometry
Authors: Ensley J. F. , Maciorowski Z. , Pietraszkiewicz H. , Hassan M. , Kish J. , Al‐Sarraf M. , Jacobs J. , Weaver A. , Atkinson D. , Crissman J. 
Published: August 2005

AbstractIntense interest in advanced squamous cell cancers of the head and neck (SCC of H&N) has resulted from the recent progress made in tumor responses with chemotherapy and radiotherapy. Unfortunately, the response patterns and clinical outcome of such patients are not adequately predicted on an individual patient basis using clinical parameters or conventional morphology. The study of flow cytometrically determined cellular parameters in such tumors is therefore of interest, but is hindered by inadequate tumor preparative technology. The previous article (10) in this journal describes the use of a murine SCC tumor, LC12, which was employed for comparative testing and determination of optimum techniques of preparation for this tumor. This report describes the application of these techniques to 144 specimens of human SCC of H&N. The mean total yield for these specimens is 7.4 × 107 cells/g of tissue. The mean viable enzymatic yield (3.3 × 107 cells/g) was higher than the mean viable mechanical yield (2.0 × 107 cells/g) except when lymph nodes were the source of the specimen (5.4 × 107 cells/g). The mean dye exclusion viability from enzymatically dissociated specimens were above 90%. Significant aneuploidal subpopulation losses were evident in mechanically dissociated and enuclated specimens. 65% of the enzymatically dissociated specimens were successfully cultured with a mean cloning efficiency of 2.1 × 10−3. Preparative techniques derived from comparative testing with a murine standard tumor have been successfully applied to 144 specimens of SCC of H&N with resultant high yields and excellent viability. Technical problems detected during the preliminary testing with LC12 were confirmed in the human tumors.
Flow cytometric analysis and sorting of plant chromosomes from Petunia hybrida protoplasts
Authors: Conia J. , Bergounioux C. , Perennes C. , Muller P. , Brown S. , Gadal P. 
Published: August 2005

AbstractA method to obtain a high metaphase index and therafter a plant chromosome suspension is described for Petunia hybrida (2n = 14). Mesophyll protoplast cultures have been used, giving easily disrupted cell walls and a high percentage of dividing cells after 42 h. On 2.5 mM colchicine‐treated cells, metaphase indexes reaching 10% were routinely obtained. The lysis medium in which the protoplast‐derived cells were disrupted was a simplified culture medium. After chromosome release, samples were stained with Hoechst 33342 dye and analysed by flow cytofluorometry. The histogram of fluorescence intensities included three peaks of metaphase chromosomes and a duplication of this flow karyotype provoked by “monochromatid chromosome.” This interpretation was established after flow sorting; micronuclei could also be observed and sorted. Of the 7 chromosomes, only the largest formed a distinct peak while the others were incompletely resolved, due to the similar DNA content of various chromosomes. Model distributions of Petunia hybrida chromosomes have been computed according to the relative chromosome length. The theoretical histograms indicated that low variability is indispensable for resolving distinctive chromosome peaks. The experimental flow karyotype was consistent with one of the models having CV of 2.5%.