bluuebunny/crossref_metadata_2025_split huggingface dataset:
Measurement of transient cDNA expression in mammalian cells using flow cytometric cell analysis and sorting
Authors: Rice Glenn C. , Pennica Diane , Borree James A. , Williams Steven R. 
Published: August 2005

AbstractGeneralized methods for quantitative and sensitive measurement of transient cDNA expression in mammalian cells using flow cytometry (FCM) are described. The techniques are applicable to a wide variety of cDNAs encoding intracellular or cell surface protein products through the use of immunofluorescenceor nonimmunofluorescence‐based detection methods. The methods illustrated have been optimized for sensitive detection of transfectants and efficient recovery of the encoding plasmids from the sorted cells. Expression levels and heterogeneities were compared using four methods of DNA transfer in addition to description of a novel method to optimize single copy transfer probabilities by multiparameter analysis. The overall sensitivities are compared by reconstruction and molecular cloning experiments to other methods of selection, such as immunoselection by panning. Through the measurement of multiple heterologous products per cell, or the measurement of multiple epitopes or binding sites per heterologous protein, expression levels on a single cell basis can be measured and correlated with other endpoints for various purposes. The ability to detect and recover rare clones based on a number of single and multiparameter selection criteria should significantly extend the use of transient mammalian cDNA expression methods for applications involving novel FCM‐based reporter cDNA assays and for cloning certain rare surface‐bound or secreted proteins using FCM.
Nuclear polymorphism and nuclear size in precarcinomatous and carcinomatous lesions in rat colon and liver
Authors: Zusman I. , Kozlenko M. , Zimber A. 
Published: August 2005

AbstractThe role of nuclear polymorphism and nuclear size in an analysis of the differences between colon and liver tumors in rats as well as in an analysis of hepatic dysplasia was studied. It was shown that colon tumors which developed following treatment of animals with a direct carcinogen alone or with a carcinogen followed by secondary bile acid were characterized by low incidence of nuclear polymorphism and by an increased nuclear size in epithelial cells. Metastatic liver tumors in rats with colon tumors were characterized by a high value for the coefficient of nuclear form polymorphism and by a significant decrease in nuclear size. Hepatic dysplasia which developed as a result of prolonged treatment with secondary bile acids was characterized by high rate of nuclear form polymorphism and by a significant increase in nuclear size. The obtained results suggest that nuclear polymorphism is dependent upon the type of tissue or organ involved in the cancerous transformation and that it may have significance as a diagnostic marker of precarcinomatous and carcinomatous lesions of digestive organs only when used in combination with other analyses.
Single‐ and double‐stranded RNA measurements by flow cytometry in solid neoplasms
Authors: El‐Naggar Adel K. , Batsakis John G. , Teague Kim , Garnsey Lisa , Barlogie Bart 
Published: August 2005

AbstractWe investigated the ability of single‐and double‐stranded RNA measurements to discriminate between neoplastic and non‐neoplastic solid tissues. Sixty‐one solid nonhematopoietic neoplasms, 10 reactive non‐neoplastic lesions, and 26 normal tissue samples were the test materials. Single‐stranded ribonucleic acid (s‐RNA) levels and double‐stranded ribonucleic acid (ds‐RNA) excess in these specimens were defined in relationship to normal human lymphocytes. The mean s‐RNA index in normal, reactive, benign, and diploid and aneuploid malignant tissue samples was 0.90, 1.54, 1.9, 1.2, and 2.2, respectively. For ds‐RNA, the mean excess level for normal, reactive, benign, and diploid and aneuploid malignant specimens was 8.5%, 18.5%, 51.0%, 36.0%, and 41.3%, respectively. No statistical differences in s‐RNA level were found between non‐neoplastic and neoplastic tissue samples. A significant difference in ds‐RNA excess was found between non‐neoplastic and benign, and diploid and aneuploid malignant neoplastic tissue (P<0.001). The specificity of s‐RNA level and ds‐RNA excess was 94.4% and 100%, and the sensitivity was 29.5% and 67.2%, respectively. Notably, ds‐RNA determinations identified 70.0% of the diploid neoplastic samples, in contrast to 20% by s‐RNA. Our preliminary data suggest that ds‐RNA may be a useful parameter and may complement DNA ploidy in identification of solid neoplasms, especially if the yield of intact cells is improved.
Comparison of gaussian and t‐distribution densities for modeling fluorescence dispersion in flow cytometric DNA histograms
Authors: Lampariello Francesco , Sebastiani Giovanni , Cordelli Eugenia , Spanò Marcello 
Published: August 2005

AbstractIn this paper, we have analyzed, on an experimental basis, the behaviour of the standardized t‐distribution with three degrees of freedom versus the gaussian model, when employed for describing the fluorescence dispersion effect in flow cytometric DNA measurements. The comparison has been performed by employing two different objective criteria based on a computer analysis for fitting DNA histograms and on a parameter related to the peak distribution shape. The results indicate that, when the peak variance is not particularly small, the two models are substantially equivalent. In the case of experimental data with very sharp peaks, the t‐distribution performs better than the gaussian density.
Coincidence in high‐speed flow cytometry: Models and measurements
Authors: Keij Jan F. , Rotterdam Ab Van , Groenewegen Ad C. , Stokdijk Willem , Visser Jan W. M. 
Published: August 2005

AbstractIn flow cytometry, the coincident arrival of particles becomes a major problem when high sample rates are required. For the development of our high‐speed photodamage flow cytometer (ZAPPER), it was of importance to understand the behavior of cells at flow rates of around 50,000–250,000 event/s. We developed and compared two models that describe the relation between the real cell rate and the detectable single cell rate. Both the Computer Simulation model and the Input/ Output Device model show distinct optima for the cell rate. The models were compared to measurements performed on the ZAPPER‐prototype. Fits of the two models to the experimental data were excellent for cycle times of 4 and 15 μs and acceptable for a 2 μs cycle time. A third model (Mercer WB, Rev. Sci. Instr. 37:1515–1521,1966) could be fitted to the experimental data, after the proportionality constant k was adapted to the experimental data. At a yield of detectable single cells of 70%, the maximum cell rates are 180,000, 100,000, and 40,000 cells/s for cycle times of 2, 4, and 15 μs, respectively. Based on these results we can now select an optimal cell rate for analysis and sorting based on criteria such as accepted cell loss. In addition, the advantages of reducing the cycle time can now be evaluated with respect to the costs of that modification.
Improved prognostic impact of S‐phase values from paraffin‐embedded breast and prostate carcinomas after correcting for nuclear slicing
Authors: Kallioniemi Olli‐ P. , Visakorpi Tapio , Holli Kaija , Heikkinen Asko , Isola Jorma , Koivula Timo 
Published: August 2005

AbstractNuclear debris may significantly interfere with the analysis of S‐phase fraction (SPF) from paraffin‐embedded tumors. We used a background subtraction algorithm to compensate for the effects of slicing of tumor cell nuclei during preparation of paraffin‐embedded specimens. DNA histograms were analyzed from 88 node‐negative breast and from 78 prostatic carcinomas. Median SPFs corrected for nuclear slicing were lower than un‐corrected ones in both breast cancer (7.6% vs. 5.7%) and prostate cancer (6.7% vs. 4.2%). The median SPF value in each group was used as a cut‐off point in survival studies. As compared with the uncorrected SPFs, corrected SPF levels resulted in a more significant survival difference between breast cancer patients with above and below median SPF (p = 0.0014 vs. p = 0.014) and in a higher relative risk (RR) of death (4.5 vs. 3.1). The same was true for prostate cancer survival (p < 0.0001 vs. p = 0.002) and RR (5.3 vs. 3.1). Compared with the exponential background subtraction method, the sliced nuclei correction was more reproducible and could be applied in all evaluable histograms without the risk of overcompensation. In conclusion, our results support the use of background correction with the sliced nuclei model in DNA flow cytometric studies of archival tissues.
Ki‐67 labeling in postmitotic cells defines different Ki‐67 pathways within the 2c compartment
Authors: Manoir Stanislas Du , Guillaud Philippe , Camus Emmanuel , Seigneurin Daniel , Brugal Gérard 
Published: August 2005

AbstractSimultaneous quantification of DNA and Ki‐67 proliferation‐associated antigen was performed using fluorescence image cytometry. In the MCF‐7 cell line, the Ki‐67 antigen content increases during the cell cycle, and its intranuclear distribution pattern varies. Quantitative evolution of Ki‐67 content as a function of nuclear area makes it possible to define several pathways followed by cells going through the 2c compartment. 1) In some cells, the amount of Ki‐67 antigen remains constant during G1 (Ki‐67 stable pathway), and a characteristic speckled pattern can be observed. 2) In the larger fraction of cells analyzed, there is a postmitotic decrease in the Ki‐67 (Ki‐67 decrease pathway) content. In this pathway, labeling is located in the nucleoplasm in small nuclei, is located in nucleoli in intermediate‐sized nuclei, and is absent from larger nuclei (G0). A progressive increase in Ki‐67 content (Ki‐67 increase pathway) was observed from intermediate‐sized nuclei to S phase nuclei. From these results, we hypothesize that the Ki‐67 stable pathway is the G1 phase of newly formed cells going directly to S phase in local optimal conditions of growth and that Ki‐67 decrease pathway and Ki‐67 increase pathway correspond to cells whose progression to S phase is regulated by extracellular factors.
Clinical applications of cytometry: 5th annual meeting
Authors: Giorgi J. V. , Cram L. S. , Parker J. W. , Dressler L. , Kidd P. , La Via M. F. 
Published: August 2005

AbstractThe 5th Annual Clinical Applications of Cytometry meeting was held September 12–15, 1990 in Charlston, SC. The theme which emerged repeatedly throughout the meeting was the need to take full advantage of the quantitative power of cytometry to provide the most useful clinically relevant diagnostic and prognostic information. Greater quantitative power is based on careful and reproducible standards and quality control. The same principles, albeit kwith somewhat different approaches, apply to cell surface immunofluorescence analysis, DNA measurements, and image cytometry assessments. Monoclonal antibody probes against aoncogenes, others against lymphokines within the Golgi, and a novel fluorogenic substrate designed to quantitate the activity of a mitochondrial enzyme were exciting developments described at the meeting.
Single beam optical trapping integrated in a confocal microscope for biological applications
Authors: Visscher K. , Brakenhoff G. J. 
Published: August 2005

AbstractConfocal microscopy is very useful in biology because of its three dimensional imaging capacities and has proven to be an excellent tool to study the 3D organization of, for instance, cell structures. This property of confocal microscopy makes it also very suitable for observation during guidance of the three dimensional manipulation of single cells or cell elements. Therefore we decided to integrate a confocal microscope and a single beam optical manipulator into a single instrument. The advantage of optical manipulation over mechanical techniques is that it is non‐invasive and therefore may be applied on living (micro‐) organisms and cells. The creation of an effective single beam optical trap requires the use of a high numerical aperture (N.A.) objective to focus the laser beam. In this paper we briefly discuss the vertical or axial force exerted on a sphere in a single beam trap. The axial force on a sphere placed on the optical axis, caused by reflection and refraction, is calculated applying a electromagnetic vector diffraction theory to determine the field distribution in the focal region. One of the results is that the particle also experiences a vertical trapping force towards the focusing lens when it is in the strongly convergent part of the field in addition to the known negative signed trapping force in the divergent part of the field. Further we describe an instrumental approach to realize optical trapping in which the optical trap position is controlled by moving the focusing objective only. At present we have demonstrated the practical use of the presented optical trap control on the spatial x, y, z manipulation of micro‐sized polystyrene spheres (1–7 μm) and of yeast cells.
Compliance of bacterial polyhooks measured with optical tweezers
Authors: Block Steven M. , Blair David F. , Berg Howard C. 
Published: August 2005

AbstractIn earlier work, a single‐beam gradient force optical trap (“optical tweezers”) was used to measure the torsional compliance of flagella in wild‐type cells of Escherichia coli that had been tethered to glass by a single flagellum. This compliance was nonlinear, exhibiting a torsionally soft phase up to 180°, followed by a torsionally rigid phase for larger angles. Values for the torsional spring constant in the soft phase were substantially less than estimates based on the rigidity determined for isolated flagellar filaments. It was suggested that the soft phase might correspond to wind‐up of the flagellar hook, and the rigid phase to wind‐up of the stiffer filament. Here, we have measured the torsional compliance of flagella on cells of an E. coli strain that produces abnormally long hooks but no filaments. The small‐angle compliance of these cells, as determined from the elastic rebound of the cell body after wind‐up and release, was found to be the same as for wild‐type cells. This confirms that the small‐angle compliance of wild‐type cells is dominated by the response of the hook. Hook flexibility is likely to play a useful role in stabilizing the flagellar bundle.
An improved hedley method for preparation of paraffin‐embedded tissues for flow cytometric analysis of ploidy and S‐phase
Authors: Heiden Thomas , Wang Naining , Tribukait Bernhard 
Published: August 2005

AbstractA modification of the Hedley‐method for flow cytometric DNA analysis of paraffin‐embedded tissues is presented. Dewaxed and dehydrated tissue from paraffin blocks was incubated with subtilisin Carlsberg (pronase, Sigma protease XXIV) and then stained directly without washing and centrifugation. The loss of material was minimized, which was advantageous, for example, for the analysis of core‐biopsies, and all measured samples showed extremely low frequencies of clumped cell nuclei. This made it easier to detect polyploid nuclei and even rare nuclei of high ploidy could be identified. S‐phase analyses were more precise, since the background originating from clumped debris particles was very low. The improved method was applied to the estimation of frequencies of high‐polyploid nuclei found in various diploid, tetraploid, and aneuploid human myosarcomas of the uterus.
Flow cytometric assay for phagocytosis of human monocytes mediated via Fcγ‐receptors and complement receptor CR1 (CD35)
Authors: Andoh Akira , Fujiyama Yoshihide , Kitoh Katuyuki , Hodohara Keiko , Bamba Tadao , Hosoda Shiro 
Published: August 2005

AbstractWe developed a simple flow cytometric assay for phagocytosis by human monocytes that is mediated via Fcγ receptors and the complement receptor CR1 (CD35), using fluorescent latex beads carrying IgG and complement components C4b and C3b. To prepare fluorescent latex beads carrying IgG(BA), BSA‐coated latex beads (B) were incubated with diluted rabbit anti‐BSA IgG. To bind complement components, BA‐particles were incubated with whole human serum pretreated with K‐76 monocarboxylic acid (K‐76COOH). K‐76COOH inhibits the activities of C5 and factor I (12,13), resulting in the deposition of C1\documentclass{article}\pagestyle{empty}\begin{document}$ \overline{4{\rm b},2{\rm a,3b}} $\end{document} on BA‐particles (BAC1\documentclass{article}\pagestyle{empty}\begin{document}$ \overline{4{\rm b},2{\rm a,3b}} $\end{document}). Further incubation of BAC1\documentclass{article}\pagestyle{empty}\begin{document}$ \overline{4{\rm b},2{\rm a,3b}} $\end{document} with EDTA‐GVB at 37°C gave particles carrying IgG and C4b,C3b (BAC4b,3b). The C3 fragment, C3b, was confirmed to present on BAC1\documentclass{article}\pagestyle{empty}\begin{document}$ \overline{4{\rm a},2{\rm a,3b}} $\end{document} particles by SDS‐PAGE and immunoblot, and these particles were calculated to have approximately 25,000–30,000 C3b molecules per particle. To evaluate the particle attachment, the phagocytic assay was performed with 3 μM cytochalasin D treated cells. The percent cells with ingested particles and the number of ingested particles/100 cells for 60 min were estimated, being 5.1% and 5.4 for B, 12.3% and 26.7 for BA, 42.5% and 108.7 for BAC4b,3b, and 42.6% and 112.5 for BAC1\documentclass{article}\pagestyle{empty}\begin{document}$ \overline{4{\rm b},2{\rm a,3b}} $\end{document}, respectively.
Attachment of A172 human glioblastoma cells affects calcium signalling: A comparison of image cytometry, flow cytometry, and spectrofluorometry
Authors: Szöllösi Jànos , Feuerstein Burt G. , Hyun William C. , Das Manoj K. , Marton Laurence J. 
Published: August 2005

AbstractThe intracellular free calcium concentration ([Ca2+]i) of indo‐1 loaded A172 human glioblastoma cells stimulated by platelet‐derived growth factor (PDGF) was studied in cell suspensions by flow cytometry and spectrofluorometry and in confluent monolayers by laser image cytometry and spectrofluorometry. With all three techniques, the percentage of responsive cells, peak [Ca2+]i, and the duration of response were directly related, and the delay time was inversely related to PDGF dose. The maximum response occurred at a PDGF concentration of about 20 ng/ml. Basal and peak [Ca2+]i did not differ significantly from method to method even though different calibration procedures were used. Cells in suspension monitored by both spectrofluorometry and flow cytometry displayed significantly shorter calcium responses than attached cells. This did not appear to be a direct effect of trypsinization. Spectral analysis of indo‐1 in cytoplasm, 40% glycerol, and aqueous solutions showed significant differences in the isosbestic point and quantum efficiency. Calibration of [Ca2+]i with spectrofluorometry is more accurate using the ratio of fluorescence intensities than the fluorescence intensities measured at either 405 or 485 nm.
Simple method for quantification of fast plasma membrane movements
Authors: van Larebeke Nicolas A. F. , Bracke Marc E. , Mareel Marc M. 
Published: August 2005

AbstractWe present a method for the quantification of the fast plasma membrane movements that are involved in ruffling, blebbing, fast shape change, and fast translocation. The method is based on the Kontron Vidas image analysis computer program. Video images from cells viewed through an inverted microscope were transmitted to the computer. The procedure was as follows: 4 consecutive video images were averaged (image1); 28 s later a second set of 4 video images was averaged (image 2); image 2 was subtracted from image 1 and the grey level of each pixel of the resulting image was increased with 128 grey level units, resulting in the subtraction image, showing a uniform grey background speckled with brighter and darker spots corresponding to areas of movement. These spots were discriminated and turned into white objects against a black background. Interactive editing was used to delete artefacts that resulted from floating debris. The total area of the discriminated objects was measured, and the parameter motile area in μm2 per cell was calculated. We have applied our method to the study of motility induced in epithelial cell lines by the tumor promoter 12‐Otetradecanoyl‐phorbol‐13‐acetate and by epidermal growth factor.
Invasive epithelial cells show more fast plasma membrane movements than related or parental non‐invasive cells
Authors: van Larebeke Nicolas A. F. , Bracke Marc E. , Mareel Marc M. 
Published: August 2005

AbstractFast plasma membrane movements (FPMM) are involved in ruffling, blebbing, fast shape change, and fast translocation. A simple method for the quantification of FPMM was used to study the relation between FPMM and invasive capacity in five pairs of invasive and noninvasive variants from four different epithelial cell types. The human mammary cell line MCF‐7/6, the ras‐transformed dog kidney cell line ras‐MDCK, the ras‐transformed mouse mammary gland cell lines NM9‐ ras‐12 and NM‐f‐ras‐TD, and spontaneously transformed late passage mouse lens explant MLE cells, all of which were invasive in vitro, showed more FPMM in our measurements and displayed more ruffling activity on timelapse video films than the related or parental MCF‐7/AZ, MDCK‐3, NM9, and NM‐f cell lines and early passage MLE cells, none of which were invasive. Interestingly, induction of invasive capacity in MCF‐7/AZ cells by retinoic acid was accompanied by an increase in FPMM, but speed of translocation was not increased. Together these observations support the hypothesis that a certain level of FPMM is a prerequisite for invasive capacity.
Utility of light scatter in the morphological analysis of sperm
Authors: Zucker Robert M. , Perreault Sally D. , Elstein Kenneth H. 
Published: August 2005

AbstractWe were able to differentiate the morphologically diverse sperm nuclei of four animal species by using an Ortho flow cytometer to detect the forward light scatter from a red (helium‐neon) laser. Cytograms depicting the axial light loss and forward red scatter signals revealed unique, but reproducible, sigmoid distributions that reflected not only interspecies differences in shape and size, but variations in particle refractive index and orientation within the flow cell at the time of analysis. Consequently, we were able to use regional gating of the light scatter cytogram to minimize the influence of orientation on the resolution of the fluorescence signal. We also observed that sperm enlarging as a result of chemically induced decondensation exhibit over time a biphasic shift (increase, then decrease) in light scatter at a species‐dependent rate. These results suggest that, without any special adaptations to the flow cytometer, light‐scatter parameters can be used to discriminate morphologically different sperm, to enhance the resolution of fluorescence measurements that may otherwise be confounded by variability in radial orientation, and to detect alterations in the rate of a biochemical/biophysical process such as decondensation.