bluuebunny/crossref_metadata_2025_split huggingface dataset:
DNA flow cytometric analysis of mouse seminiferous epithelium
Authors: Toppari Jorma , Bishop Philippe C. , Parker John W. , Dizerega Gere S. 
Published: August 2005

AbstractIn order to provide a basis for quantitative studies of murine spermatogenesis, we performed a DNA flow cytometric analysis on the mouse seminiferous tubules isolated at defined stages of the epithelial cycle by transillumination‐assisted microdissection. Accurate stage identification was performed by examining spermatids in the adjacent tubule segments by phase‐contrast microscopy. For flow cytometry, suspension of nuclei of spermatogenic cells was obtained by detergent treatment of isolated seminiferous tubules, and fresh samples were stained with propidium iodide.DNA histograms of the 12 stages of the mouse seminiferous epithelial cycle varied in a stage‐specific manner. DNA histograms of stages I‐VIII of the cycle were characterized by a hypofluorescent haploid peak, the location of which changed with the decreasing DNA dye (propidium iodide)‐binding capacity of elongated spermatids. The absence of the hypohaploid peak and the high ratio of the cells with 4C amount of DNA to the cells with 1C amount of DNA characterized stages IX‐XI of the cycle. Stage XII showed a high 2C peak, owing to a large population of secondary spermatocytes arisen from the first meiotic division. By using fluorescent beads as an internal volume standard cell numbers in defined stages were determined. These data provide a basis for quantitative studies of mouse spermatogenesis.
Flow cytometric detection of ribosomal RNA in suspended cells by fluorescent in situ hybridization
Authors: Bauman J. G. J. , Bentvelzen P. 
Published: August 2005

AbstractA method using flow cytometry and fluorescent in situ hydridization (ISH) to detect RNA in cells is described. L1210 murine leukemia cells were fixed with 1% formaldehyde in HEPES buffered Hank's balanced salt solution (HH) followed by 70% ethanol. Endogenous RNAses were blocked by diethylpyrocarbonate treatment. Single‐stranded sense and antisense RNA probes, labeled with biotin‐11‐UTP, were transcribed from a 2.1 kb 28S ribosomal RNA (rRNA) gene fragment subcloned into the pGEM2 plasmid. For good results, it was essential that the probes were degraded to 100–150 nucleotides before use. Hybridization was performed at 45°C in 50% formamide, 5 × SSC, 0.5% SDS. Hybrids were detected with streptavidin‐FITC by flow cytometry. Antisense rRNA probe signal was 100 times higher than the background. The hybrids were largely resistant to RNAse and melted at high temperature. The sense probe also gave a signal (5 times background), which was not RNAse resistant and was attributed to the presence of internal inverted repeats in the ribosomal RNA. When sufficient background reduction can be achieved, it is expected that as few as ten mRNA molecules per cell can be detected with the fluorescent in situ hybridization method.
Correction of cellular autofluorescence in flow cytometry by mathematical modeling of cellular fluorescence
Authors: Corsetti James P. , Sotirchos Stratis V. , Cox Christopher , Cowles Judith W. , Leary James F. , Blumburg Neil 
Published: August 2005

AbstractA method for the correction of background fluorescence in flow cytometry with special relevance to the quantitation of low levels of cellular surface membrane antigens is presented. The method is based on the mathematical modeling of cellular fluorescence distributions of background fluorescence (autofluorescence control or irrelevant antibody control) and total fluorescence (positively stained cells). Algorithms based on two models and utilizing only the routinely available background and total fluorescence histograms are developed and implemented in computer programs. These allow estimation of the fluorescence histogram corresponding exclusively to immunofluorescence staining of the cell surface antigen of interest. Thus, the correction of background fluorescence is effected solely with software processing of routinely available data; no additional hardware or parameter determinations are necessary.Two models were chosen to be physically plausible and to represent extremes in correlation between background and probe fluorescence. Extremes were chosen to assess the solution dependence on model and to provide bounds to the actual solution when no information on correlation is available. Results are presented for both computer simulations and for an actual assay of the CR1 complement receptor on human erythrocytes to test and illustrate the technique. Alternatively, data can be tested assuming a particular model to explore the relationship, if any, between specific and nonspecific fluorescence.
Comparison between flow cytometry and fluorometry for the kinetic measurement of membrane fluidity parameters
Authors: Collins James M. , Grogan W. McLean 
Published: August 2005

AbstractSteady‐state fluorescence polarization measurements obtained with a flow cytometer were compared with those obtained with an SLM subnanosecond fluorometer. Measurements were made over time after exposure of HeLa cells to the membrane probe 1,6‐diphenyl‐1,3,5‐hexatriene (DPH), 1‐[4‐(trimethylamino) phenyl]‐6‐phenyl‐1,3,5‐hexatriene (TMA‐DPH), or 12‐(9:anthroyloxy) stearate (12‐AS). After 1 min, anisotropy values of 0.28 (DPH), 0.28 (TMA‐DPH), and 0.21 (12‐AS) were obtained. Thereafter, the anisotropy of DPH‐ and 12‐AS‐labelled cells rapidly decreased (0.18 and 0.12 after 5 min), while that of TMA‐DPH‐labelled cells changed only slightly (0.27 after 30 min), suggesting that DPH and 12‐AS, unlike TMA‐DPH, do not remain anchored in the HeLa plasma membrane, but translocate to more fluid environments inside the cell. These suggestions were confirmed by visual observation with fluorescence microscopy. There was no significant difference between the results obtained with the flow cytometer and those obtained with the fluorometer.
Characterization by flow cytometry of fluorescein‐methotrexate transport in chinese hamster ovary cells
Authors: Assaraf Yehuda G. , Seamer Larry C. , Schimke Robert T. 
Published: August 2005

AbstractWe have studied by flow cytometry the transport of fluorescein‐methotrexate in Chinese hamster ovary cells. Fluorescein‐methotrexate appears to enter cells via a mechanism different from the carrier‐mediated system for methotrexate. This conclusion is supported by the following observations: (1) Fluorescein‐methotrexate is transported equally well into normal and mutant cells defective in the in ward methotrexate uptake. (2) Folic acid and its reduced states, which competitively inhibit methotrexate uptake, do not alter fluorescein‐methotrexate transport. (3) Fluorescein‐methotrexate accumulation exhibits a low temperature coefficient (Q10 = 1.6) compared with the influx of methotrexate (Q10 = 6–8). (4) Initial rates of fluorescein‐methotrexate uptake are concentration dependent but are not saturable. (5) Fluorescein‐methotrexate uptake is very slow and reaches steady state after 8 h, whereas at an equimolar concentration methotrexate reaches saturation after 20 min. (6) Initial influx rates of fluorescein‐methotrexate are not affected by the presence of methotrexate. (7) Sulfhydryl‐reactive mercurials, which block methotrexate transport, do not reduce fluorescein‐methotrexate influx, but rather stimulate it. Thus, based on the nonsaturability of fluorescein‐methotrexate inward transport, its low temperature coefficient, and lack of inhibition with structural analogs, we conclude that fluorescein‐methotrexate is accumulated in hamster cells by a passive diffusion process.
Comparison of fresh, ethanol‐preserved, and paraffin‐embedded samples in DNA flow cytometry
Authors: Alanen Kalle A. , Klemi Pekka J. , Joensuu Heikki , Kujari Harry , Pekkala Erkki 
Published: August 2005

AbstractFresh, ethanol‐preserved, and formalinfixed and paraffin‐embedded samples taken from the same part of 15 human tumors, and from one normal spleen and one pancreas were analyzed for nuclear DNA content by flow cytometry. The coefficient of variation (CV) values of the Gl peaks were smaller in the fresh than in the other samples (P < 0.001). The DNA ploidy of the tumors was the same in all types of samples. The DNA indices (DIs) measured from either ethanol‐preserved or formalin‐fixed tissue correlated strongly with those obtained from fresh tissue (P < 0.001), although they tended to be somewhat smaller in the fresh samples. The S‐phase fractions measured from all types of samples were of the same order of magnitude in most cases (P < 0.001). Uninterpretable histograms were most often obtained from fresh samples. Identical DI values and rather constant S‐phase fractions were obtained from ethanol‐preserved samples stored at 4°C for up to 5 months. It is concluded that all three types of samples are suitable for the determination of DNA ploidy, DI, and S‐phase fraction and that 50% ethanol is suitable for long‐term preservation of flow cytometric samples.
Flow cytometry measurements of human chromosome kinetochore labeling
Authors: Fantes Judith A. , Green D. K. , Malloy Patricia , Sumner A. T. 
Published: August 2005

AbstractA method for the preparation and measurement of immunofluorescent human chromosome centromeres in suspension is described using CREST antibodies, which bind to the centromeric region of chromosomes. Fluorescein isothiocyanate (FITC)‐conjugated antihuman antibodies provide the fluorescent label. Labeled chromosomes are examined on microscope slides and by flow cy tometry. In both cases a dye which binds to DNA is added to provide identification of the chromosome groups. Sera from different CREST patients vary in their ability to bind to ch romosome arms in addition to the centromeric region. Flow cytometry and microfluorimetry measurements have shown that with a given CREST serum the differences in kinetochore fluorescence between chromosomes are only minor. Flow cytometry experiments to relate the number of dicentric chromosomes, induced by in vitro radiation of peripheral blood cells to the slightly increased number of chromosomes with above‐average kinetochore fluorescence did not produce decisive radiation dosimetry results.
Subdivision of S‐phase by analysis of nuclear 5‐bromodeoxyuridine staining patterns
Authors: Van Dierendonck Jan Hein , Keyzer Rob , van De Velde Cornelis J. H. , Cornelisse Cornelis J. 
Published: August 2005

AbstractAfter pulse labeling of mammalian cells in vivo or in vitro with 5‐bromodeoxyuridine (BrdUrd), followed by immunostaining with a monoclonal antibody to DNA‐incorporated BrdUrd, various intranuclear staining patterns are observed. These results are obtained in various labeling, fixation, denaturation, and staining conditions. We defined five different patterns in immunoperoxidasestained monolayers of human and rodent cancer cells and compared mean nuclear areas as measured by computerized planimetry. Furthermore, we evaluated frequency distributions of these patterns in partly synchronized cell populations and correlated these results with flow cytometric DNA histograms.The results indicate that the observed patterns reflect the spatial and temporal organization of DNA synthesis, which seems to be characterized by at least three successive stages of replication. Evaluation of these patterns may have various applications in studies on cell cycle kinetics.
Simultaneous paired analysis by flow cytometry of surface markers, cytoplasmic antigens, or oncogene expression with DNA content
Authors: Dent Georgette A. , Chantal Leglise M. , Pryzwansky Katherine B. , Ross Dennis W. 
Published: August 2005

AbstractFlow cytometry is a useful tool for measuring DNA content and differentiation as expressed by cell surface markers. We have extended this technology to measure simultaneously either surface, cytoplasmic, or nuclear antigens (particularly oncoproteins) with DNA content. Mononuclear blood cells isolated from normal subjects and HL60 leukemic cells were permeabilized and fixed in suspension utilizing 40 μg/ml lysolecithin and 1% paraformaldehyde. A range of lysolecithin concentrations in 1% paraformaldehyde was studied to optimize permeabilization of the antibodies to the cell interior without destroying cell integrity. The optimal concentration (40 μg lysolecithin/ml) resulted in good cell recovery with a high percentage of cells positive for surface and intracellular antigens. Cells are first stained with fluorescein isothiocyanate conjugated (FITC) anti‐myeloperoxidase (an azurophil granule enzyme), or with an anti‐c‐myc antibody and FITC goat anti‐mouse IgG F(ab')2. Cells are then incubated with RNase and stained for DNA content with propidium iodide. Alternatively, cells were stained for the cell surface markers Leu M3, OKM1, or the transferrin receptor and were then fixed and permeabilized and stained with propidium iodide. Using this method, we correlated cytoplasmic, nuclear, or cell surface antigens with cell cycle kinetics. This technique should be useful for studies of cellular differentiation and proliferation.
Detection of mRNA in flow‐sorted cells
Authors: Dunne J. F. , Thomas J. , Lee S. 
Published: August 2005

AbstractCells were sorted onto nitrocellulose filters which were saturated with a lysing cocktail designed to preferentially immobilize cellular mRNA. After washing, these filters were incubated with 32P‐labeled specific DNA probes. We used the phorbol ester/lipopolysaccharide (PMA + LPS) co‐induction of IL‐1 mRNA and CD13 expression in U937 cells to demonstrate the specificity of the technique. In addition we used the abundant expression of c‐fos in U937 to demonstrate linearity. IL‐1 beta mRNA is readily discernable autoradiographically from as few as 5,000 PMA + LPS‐induced cells sorted onto a filter. With liquid scintillation counting we demonstrate good linearity of the c‐fos quantitation over the range of 1,000 cells to 60,000 cells per filter target. The technique is easily adapted to any sorting flow cytometer and should prove useful to help correlate any flow cytometric cell phenotype with specific mRNA abundance.
Correlation of DNA content, p53, T antigen, and V antigen in simian virus 40‐infected human diploid cells
Authors: Laffin Judith , Fogleman David , Lehman John M. 
Published: August 2005

AbstractHuman diploid fibroblasts (HDF) have a finite life span in cell culture which can be extended when transformed with simian virus 40 (SV40). Flow cytometric analysis of SV40‐HDF transformation allowed DNA content changes to be correlated with the appearance, quantity, and distribution of T antigen, p53, and V antigen, three proteins associated with this process. These studies demonstrated a shift in the DNA content to tetraploidy, which was correlated with the age of the SV40‐HDF but not the time of infection. A significant increase of the epitope recognized by Pab122 to host p53 and the epitope PAb101 to SV40 T antigen occurred at the same time the tetraploid population appeared. However, an antigen reactive with SV40 V antibody was present at high levels in most of the population early after infection, but the levels declined with time. The percentage of PAb101‐T antigen‐positive cells increased more rapidly in cells infected at a late passage, and this was concomitant with the shift in DNA content to tetraploid. Analysis of the mean fluorescence of total, gated populations (G1, G2, and >G2) demonstrated that a threshold level of p53 and T antigen was reached in each compartment of the cell cycle. As the transformed phenotype appeared, a population of cells was continually released into the supernatant, and although these cells had a DNA pattern similar to the monolayer cells, the T antigen and p53 levels were 3–5 times higher in the tetraploid G2 cells.These studies correlated the expression of proteins associated with viral transformation in HDF which vary with time and shift in DNA content.
Discrimination of benign versus malignant mixed tumors of the salivary gland using digital image analysis
Authors: Layfield Lester J. , Hall Thomas L. , Fu Yao S. 
Published: August 2005

AbstractSeven benign and four malignant mixed tumors of the salivary gland, biopsied using fine‐needle aspiration, were analyzed using digital image analysis. Mean nuclear form factor, perimeter, and area were significantly increased in malignant cases. Better separation between diagnostic categories, however, was achieved by utilizing the coefficient of variation (CV) within a case rather than mean value. Form factor CV alone divided cases into nonoverlapping diagnostic categories. This quantitative analog of “pleomorphism” provided a useful marker for malignancy in mixed tumors.
Continuous bromodeoxyuridine labeling and bivariate ethidium bromide/hoechst flow cytometry in cell kinetics
Authors: Poot Martin , Schmitt Heidi , Seyschab Helga , Koehler Julia , Chen Una , Kaempf U. , Kubbies Manfred , Schindler Detlev , Rabinovitch Peter S. , Hoehn Holger 
Published: August 2005

AbstractMost techniques of flow cytometric cell cycle analysis are not capable of distinguishing the number of rounds of DNA synthesis that a cell has undergone since the start of an experiment. Continuous labeling with 5‐bromodeoxyuridine (Brd‐Urd) offers such a potential. We illustrate here that the bivariate analysis of non‐BrdUrd‐quenched ethidium bromide vs. BrdUrd‐quenched Hoechst 33258 fluorescence offers a high degree of resolution that enhances the analytical power of the technique, and that this approach can be applied to the analysis of a broad range of human and murine primary cells and established cell lines.
On fully automatic feature measurement for banded chromosome classification
Authors: Piper Jim , Granum Erik 
Published: August 2005

AbstractProcedures for fully automatic location of chromosome axis and centromere in metaphase chromosomes are described for a practical interactive chromosome analysis system that omits the usual stages of interactive axis and centromere correction. Accuracy of centromere finding and consequential determination of a chromosome's polarity, i.e., which end is which, is measured experimentally. The saving in interaction by not correcting centromeres is compared to the increase in errors at the classification stage and the consequent increase in interaction needed to correct these errors. Some previously unreported features for banded chromosome classification are described, and in particular a set of global shape features is introduced. The discrimination capability of the feature measurements is evaluated by use of simple statistics and by reference to the performance of classifiers trained with various feature subsets. Class discrimination capability of the global shape feature set is shown to be comparable to that of centromere position, a widely used local shape feature. The variability of feature measurements that might occur in data from different laboratories on account of differing tissue, preparation methods, and digitiser hardware is assessed using three data bases of G‐banded human metaphase cells. It is shown that the differences can be considerable and that appropriate feature selection and classifier training substantially improve classification performance.
A simple electronic volume cell sorter for clonogenicity assays
Authors: Freyer James P. , Wilder Mark E. , Schor Patricia L. , Coulter James , Raju Mudundi R. 
Published: August 2005

AbstractA single‐parameter electronic volume flow cell sorter that can be easily and inexpensively constructed using existing technology is described. The instrument is designed for ease and flexibility of operation, including such features as a large open area for recovering sorted cells into a variety of dishes or vessels; a remote, electrically activated fluidics system; a mechanism for heating or cooling samples during sorting; a simple arrangement for monitoring and adjusting the sorting control parameters; and an interface to a standard IBM personal computer for data acquisition, analysis, and control of the sorting windows. Several researchers in our laboratory now routinely use this sorter for plating precise numbers of cells directly into culture dishes in an aseptic manner for clonogenicity assays. The instrument can sort cells at rates of up to ∼2,000 per second with >80% sorting efficiency and no cytotoxicity. An advantage of this system is that the sorting windows can be set to exclude acellular debris and include either the entire cell volume distribution or a subset thereof. Applications of the instrument are detailed, including (1) precise cell plating for low‐dose survival studies, (2) separation of cells into age compartments, and (3) rapid inoculation of single cells into multiwell dishes for cloning studies. Advantages of this technology for cell survival studies are detailed, along with some limitations to its applicability.
Parallel processing data acquisition system for multilaser flow cytometry and cell sorting
Authors: van den Engh Ger , Stokdijk Willem 
Published: August 2005

AbstractThis report describes the data acquisition electronics for a flow cytometer. The design differs from most instruments in that the signals from a large number of detectors are processed in parallel. Each of the input channels is capable of autonomously measuring and digitizing the fluorescence signals. The digitized values that belong to one particle are collected by digital circuitry and are presented as a compact data package on a special bus. In addition to the pulse values, the data package contains a time marker, information needed for sort decisions, and an error detection code. Specially designed electronic modules that read the information from the bus can take complex multiparameter sort decisions at a very high speed. All events can also be recorded as data lists by a computer. The lists can be used to reconstruct a sort or analysis run. The raw data lists can also be reduced to kinetic curves and/or (gated) multivariate histograms. As a result of the applied scheme of parallel pulse processing, the dead time of the system is independent of the number of parameters measured and the number and time separation of the excitation beams. The instrument has a cycle time of 5 m̈s, which corresponds to a throughput rate of 2 × 105 events/s. At this rate, the incidence of correlation errors is well below 1 in 108 analyzed particles. The system has proved to be reliable and convenient to use in a variety of experiments. Its high speed and low error rate make it well suited for high‐resolution measurements, rare‐event analysis, kinetic measurements, and high‐speed cell sorting.