bluuebunny/crossref_metadata_2025 huggingface dataset:
Flow cytometric analysis of two incorporated halogenated thymidine analogues and DNA in a mouse mammary tumor grown in vivo
Authors: Pollack Alan , Terry Nicholas H. A. , Van Nguyen T. , Meistrich Marvin L.
Published: August 2005

A technique was developed for the staining of nuclei for DNA using propidium iodide, and incorporated chlorodeoxyuridine (CldUrd) and iododeoxyuridine (IdUrd) using two monoclonal antibodies that showed negligible cross‐reactivity. The mouse mammary solid tumor MCaK was labeled in vivo by intraperitoneal injection of the nucleosides. Tumor cell nuclei were stained after isolation from ethanol‐fixed solid tumor tissue and acid denaturation. The Br3 antibody, which specifically recognizes CldUrd, was applied first, followed by indirect staining with goat anti‐mouse phycoerythrin. The direct fluorescein isothiocyanate conjugate of the B44 antibody, which specifically recognizes IdUrd, was then applied. In the direct conjugate form this antibody reacted only minimally with CldUrd. The nuclei were then stained with propidium iodide. With this dye combination the coefficients of variations of the DNA histograms were consistently in the 2–4% range. Two other dye combinations were compared. The propidium iodide/phycoerythrin/fluorescein isothiocyanate dye combination was the simplest because of the compatibility with single laser flow cytometry. © 1993 Wiley‐Liss, Inc.
Cd20 (pan‐B cell) antigen is expressed at a low level on a subpopulation of human T lymphocytes
Authors: Hultin Lance E. , Hausner Mary Ann , Hultin Patricia M. , Giorgi Janis V.
Published: August 2005

Despite the previous description of the leukocyte differentiation antigen CD20 as B cell restricted, the findings reported here indicate that a small subset of human T cells expresses low levels of CD20 or a cross‐reacting antigen. Three different CD20 monoclonal antibodies (mAb), Leu16, B1, and 1F5, reacted with the T cell subset. B cells that expressed CD20 were CD20bright and constituted an average of 9.2 ± 3.3% of adult PBL. Meanwhile, T cells that expressed CD20 were CD20dim and represented 2.4 ± 1.5% of the PBL. This population may have been overlooked in previous studies due to the low level of CD20 expression per T cell and the small size of the subset in most individuals. Blocking studies indicated that CD20 mAb binding to CD3+ cells was due to the antigen‐reactive regions of the CD20 antibodies and was not a result of Fc receptor binding, or non‐specific fluorochrome or protein binding. The T cell nature of the CD20dim CD3+ cells was confirmed by the rapid rise in the intracellular calcium concentration ([Ca2+]i) of CD20dim cells observed following treatment with CD3 mAb but not following treatment with anti‐human immunoglobulin (Ig). Extensive three‐color immunophenotypic analyses indicated that CD20dim T cells were phenotypically heterogeneous and displayed a leukocyte differentiation profile that was slightly different than that of CD20− T cells. Thus, the CD20dim T cells were more likely than CD20− T cells to be γ/δ T cell antigen receptor positive (14% vs. 3.4%), CD8+ (57% vs. 33%), and CD45RO+ (82% vs. 51%); fewer were CD38+ (5% vs. 24%) or CD4+ (35% vs. 61%). Given that most differentiation antigens expressed on leukocytes participate in the functions of the cells that express them, it is possible that CD20 plays a role on the T cells on which it is expressed, although the function(s) of the CD20 molecule on T and B cells remains unknown. © 1993 Wiley‐Liss, Inc.
Flow cytometric analysis of DNA and nuclear protein in paraffin‐embedded tissue
Authors: Ciancio Gaetano , Pollack Alan , Block Norman L.
Published: August 2005

Previously we described the simultaneous quantification of DNA and nuclear protein in unfixed tissue from solid tumors. The resultant 2 parameter flow cytometric analysis has several advantages over that of DNA alone. In this report, we describe a modification of the technique for the analysis of formalin‐fixed paraffin‐embedded tissue. Paraffin‐embedded material was prepared by hydrating sections, incubating in 0.5% pepsin solution, washing, and resuspending in buffer containing nonionic detergent. The nuclei were then stained with fluorescein isothiocyanate and propidium iodide in the presence of ribonuclease. Several solid tumor tissue types have been analyzed, including breast, colon, kidney, and thymus. The best results were obtained when the initial pepsin treatment was for 1.5 h, instead of 0.5 h. Pepsin treatment for 1.5 h improved the CVs of both the DNA and nuclear protein parameters, and did not appear to reduce nuclear protein levels or to cause significant disintegration of nuclei. The DNA/nuclear protein histograms of unfixed and fixed, paraffin‐embedded tissue were similar. Since tumor nuclei typically have higher protein levels than DNA‐diploid nuclei, the technique reduces population overlapping and permits less subjective identification of DNA aneuploidy. © 1993 Wiley‐Liss, Inc.
Use of mouse hepatocytes for the flow cytometric determination of DNA levels of nuclei extracted from fresh tissue of hybrid larch (Larix × eurolepis Henry)
Authors: Wyman Janet , Guertin France , Mansour Saad , Fournier Michel , Laliberté Sylvie
Published: August 2005

A rapid and reliable method is presented to release intact nuclei from small amounts (100 mg) of fresh plant tissue. Further, an accurate and readily accessible new standard is proposed. Both techniques have potential application for many plant systems. The system chosen as a standard (inbred mouse strain Balb/C or B6/AF1 hepatocyte nuclei) contains both diploid and polyploid cells. This system was applied in the flow cytometric determination of absolute nuclear DNA values of female gametophytes and in vitro propagated shoots of hybrid larch (Larix X eurolepis Henry). The amount of DNA in 2C nuclei of in vitro grown larch is 32.48 ± 4.04 or 31.97 ± 6.14 pg/nucleus, respectively, when calculated using the mouse hepatocyte 4C or 8C nuclear peak as a reference standard. The amount of DNA in female gametophyte nuclei is 17.47 ± 1.33 pg DNA/nucleus when these haploid larch nuclei were analyzed with trout red blood cell nuclei as the standard. When hepatocyte 4C nuclei were used as a standard, the absolute value of DNA per haploid larch nucleus was estimated as 16.8 ± 0.53 pg. Plant tissue with as little as 4–6 pg DNA/nucleus up to as much as 35 pg DNA/nucleus can be tested using mouse hepatocytes as a standard while retaining an optimal sample/standard ratio. © 1993 Wiley‐Liss, Inc.
Changes in intracellular free calcium during hyperthermia: Effects of local anesthetics and induction of thermotolerance
Authors: Dynlacht Joseph R. , Hyun William C. , Dewey William C.
Published: August 2005

We wished to determine if local anesthetics (LAs) induced changes in intracellular free calcium ([fCa2+i]) that could have an effect on cell killing by hyperthermia. Flow cytometry was used to measure [fCa2+i] of mouse NIH 3T3 cells during heating at 45.5°C. In both non‐tolerant and thermotolerant cells, heating caused a rapid increase (within 1 min) in [fCa2+i] of ∼100 nM, which remained relatively constant during 25 min of continued heating; however, survival was higher in thermotolerant cells. Procaine, lidocaine, and tetracaine had no effect on survival or [fCa2+i] of cells kept at 37°C up to 25 min. Cells heated with procaine and lidocaine showed no difference in [fCa2+i] compared to cells heated without LAs but were greatly sensitized to killing. Cells heated with tetracaine became permeable to trypan blue within 10–15 min of heating. We conclude that heat sensitization by LAs does not involve changes in [fCa2+i]. Furthermore, these studies reject the hypothesis that changes in [fCa2+i] are involved in heat‐induced cell killing. © 1993 Wiley‐Liss, Inc.
Flow cytometric measurement of [Ca2+]i and pHi in conjugated natural killer cells and K562 target cells during the cytotoxic process
Authors: Van Graft Marja , Kraan Yvonne M. , Segers Ine M. J. , Radoševic Katarina , De Grooth Bart G. , Greve Jan
Published: August 2005

We describe a flow cytometric assay that enables one to follow conjugate formation between cytotoxic cells and their target cells during the cytotoxic process. In addition, the internal calcium concentration ([Ca2+]i) and internal pH (pHi) of the conjugated cells can be monitored and directly compared to the nonconjugated cells. This is achieved by labeling one cell type with the Ca2+‐specific dye Fluo‐3, while the other cell type is labeled with the pH‐sensitive dye SNARF‐1. As these fluorochromes have different emission spectra, events positive for both fluorochromes are identified as conjugates. The results show that the conjugates can be clearly distinguished from single cytotoxic cells [natural killer (NK) cells] and target cells [K562 cells, (TC)]. Upon binding, [Ca2+]i is increased in the NK cells as well as in the TC. In conjugated NK cells this increase of [Ca2+]i is temperature dependent and is followed by a decrease to a normal [Ca2+]i value later on. The [Ca2+]i in NK cells increases in 2 steps, which may be related to the binding‐and lethal hit phase. Upon conjugate formation, NK cells show a slight increase in pHi (0.2–0.3 pH units). TC do not reveal a significant change in pHi. © 1993 Wiley‐Liss, Inc.
Isolation of high quality mRNA from a discrete cell cycle population identified using a nonvital dye and fluorescence activated sorting
Authors: Church Jon G. , Stapleton Ernest A. , Reilly Brian D.
Published: August 2005

Flow cytometry (FCM) is useful for measuring DNA content as related to cell cycle position. We have extended this technology by developing a method that measures cellular DNA content using pro‐pidium iodide after permeabilization with lysolecithin. This technique maintains cell integrity such that high quality mRNA can be isolated from a sorted population. Unfixed MDA‐468 cells, a human breast cancer cell line, were temporarily permeabilized with lysolecithin. A range of lysolecithin concentrations were studied in order to optimize DNA staining but minimize alterations in cell size and integrity. Cells permeabilized with lysolecithin in PBS, were stained with propidium iodide (50 μg/ml) in the presence of 1% bovine serum albumin, 1 mM Na2EDTA in PBS. The optimal concentration (4 μg lysolecithin/ml) combined staining ∼90% of the cells for DNA, with minimal effects on cell size. Subsequently, the cells were assayed for DNA content as a measure of cell cycle position. MDA‐468 cells identified as being in G1 were sorted and collected. From these, high quality mRNA could be isolated, as judged by its ability to be in vitro translated into protein of a wide range of molecular weights. This technique should be useful for molecular studies requiring discrete cell populations based on DNA content and/or cell cycle position. © 1993 Wiley‐Liss, Inc.
Multiparameter flow cytometric analysis of a novel cytotoxin (Factor 2) induced tumor cell membrane permeability
Authors: Ni Jian , Watson James V. , Cox Hilary , Karpas Abraham
Published: August 2005

An improved twin‐probe multiparameter flow cytometric technique was applied to examine a novel cytotoxin, Factor (F2), induced tumor cell permeability. Ability to retain preloaded intracellular bis‐carboxyethyl carboxyfluorescein (BCECF, green fluorescence) and to exclude extracellular propidium (red fluorescence) was measured simultaneously with forward and right‐angle scatter. In addition to the two expected cell populations which were stained green negative, red positive (“membrane‐damaged” and “non‐viable”, Region 2), and green positive, red negative (“membrane intact” and “viable”, Region 3), a third population was seen which fluoresced neither green nor red and displayed intermediate light scatter characteristics (Region 1). K562 cells progressed from Region 3 to Region 1, and then from Region 1 to Region 2 after treatment with F2. These results suggest that sequential changes in membrane structure lead to increased permeability, first with respect to intracellular BCECF and then in turn to extracellular propidium. Flow cytometric changes caused by F2 were detectable 10 min after treatment with 2.5 U/ml of F2, and 5 min after 10 or 40 U/ml of F2. Flow cytometric analysis showed that F2‐induced tumor cell lysis and growth inhibition were accompanied by rapid alternations in tumor cell membrane permeability. Flow cytometric analysis also distinguished F2 cytotoxicity from phorbol myristate acetate (PMA) associated cytotoxicity to K562 cells and determined that F2 produced spontaneously or induced by PMA and/or ciprofloxacin had a similar ability to induce tumor cell membrane permeability change. © 1993 Wiley‐Liss, Inc.
DNA content parameters of paraffin‐embedded soft tissue sarcomas: Optimization of retrieval technique and comparison to fresh tissue
Authors: Zalupski Mark M. , Maciorowski Zosia , Ryan James R. , Ensley John F. , Hussein Mohamed E. , Sundareson Alistair S. , Baker Laurence H.
Published: August 2005

DNA content analysis of formalin fixed paraffin embedded (FFPE) tissue permits determination of the influence of DNA content on the prognosis in cohorts of patients for whom the clinical outcome is known. Of key importance in such an analysis is the accuracy of DNA content determination. Variations in the quality of DNA histograms from FFPE tissues of different types prompted a comparative evaluation of the preparative methodology of FFPE soft tissue sarcomas for DNA flow cytometry. Following deparafflnation and rehydration of fixed tissue, and prior to fluorochrome staining, tissue blocks of 15 DNA aneuploid soft tissue sarcomas were subjected to repeated experimental (time × concentration) enzyme exposures. The goal of these studies was to define the optimal tissue specific retrieval technique with the coefficient of variation, maintenance of DNA aneuploidy, and DNA index as endpoints. After optimizing the technique, the DNA content of 50 soft tissue neoplasms derived from FFPE specimens was compared to the corresponding fresh surgical tissue. The observed 14 percent error rate in the determination of DNA ploidy status suggest limited utility for FFPE tissue in prospective therapeutic trials of Soft tissue sarcoma. © 1993 Wiley‐Liss, INc.
Flow cytometric analysis of xenobiotic metabolism activity in isolated rat hepatocytes
Authors: Black Kurt A. , Novicki Deborah L. , Vincent Jack L. , Smith Gary J.
Published: August 2005

Flow cytometry offers great potential for the study of xenobiotic metabolism in intact cells. We explored this application by the use of ethoxyfluorescein ethyl ester (EFEE) and isolated rat hepatocytes, a classic system for studying such reactions. EFEE is only weakly fluorescent and it diffuses freely into viable cells, where it is metabolized to fluorescein by a process dependent upon mixed‐function oxidase activity. In the current study, viable hepatocytes were first identified by flow cytometric assessment of fluorescein diacetate staining. The viable subpopulation was also identifiable on the basis of forward and right angle light scattering properties alone, and it was in this fraction that EFEE metabolism was measured. Metabolism of EFEE to fluorescein was quantified by flow cytometry. SKF 525A, α‐naphthoflavone, and metyrapone, classic inhibitors of mixed‐function oxidation, each inhibited the metabolism of EFEE. These results demonstrate the potential of EFEE for use in flow cytometric studies of drug metabolism, such as in multiparameter mechanistic assays of cellular xenobiotic metabolism and toxicity, and in the isolation by fluorescence‐activated cell sorting of subpopulations which differ in this activity. © 1993 Wiley‐Liss, Inc.
Flow cytometric evaluation of human basophils
Authors: Gane Pierre , Pecquet Catherine , Lambin Patrick , Abuaf Nissen , Leynadier Francisque , Rouger Philippe
Published: August 2005

The expression of CD45 and IgE cell surface antigens on human leucocytes was studied by flow cytometry. More than 80% of sorted cells that expressed low CD45 (CD45dim) and high IgE (IgEbright) antigen site density were identified as basophils. Immunomagnetic depletion of the CD45dim‐IgEbright cell subset by a biotin‐coupled anti‐IgE antibody and streptavidin‐coated magnetic beads was greater than 90%, and more than 80% of cells binding significant numbers of beads exhibited the morphological characteristics of basophils. Interestingly, when the cell staining was performed in the presence of calcium and magnesium, we observed a significant increase of CD45 and an equivalent decrease of IgE cell surface expression, as well as an IgE concentration dependent diminution of the number of CD45dim‐IgEbright cells. © 1993 Wiley‐Liss, Inc.
Demonstration of an S phase population of cells without DNA synthesis generated by cisplatin and pentoxyifylline
Authors: Perras James P. , Ramos Reinaldo , Sevin Bernd‐Uwe
Published: August 2005

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Flow cytometric measurement bof RNA synthesis using bromouridine labelling and bromodeoxyuridine antibodies
Authors: Østrup Jensen Peter , Larsen Jacob , Christiansen Jette , Larsen Jørgen K.
Published: August 2005

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The homing cursor: A tool for three‐dimensional chromosome analysis
Authors: Houtsmuller A. B. , Smeulders A. W. M. , van der Voort H. T. M. , Oud J. L. , Nanninga N.
Published: August 2005

When studying the three‐dimensional shape of prophase chromosomes (or any other tubular structure), it is useful to represent these structures as a string of three‐dimensional Cartesian coordinates along the medial axis. This procedure was automated in order to limit the number of human interactions and to improve reproducibility. In this paper the design, implementation, and validation of the automated method is presented. From the data presented it can be concluded that the cursor algorithm provides an objective and therefore reproducible method to trace the medial axes of prophase chromosomes automatically. This method could allow a more extensive understanding of the (changes in) chromosome organisation throughout the cell cycle, its relation to cell function, and the complex process of chromosome condensation. © 1993 Wiley‐Liss, Inc.
Derivative domain fitting: A new method for resolving a mixture of normal distributions in the presence of a contaminating background
Authors: Moore Dan H. , Gray Joe W.
Published: August 2005

Derivative domain least squares analysis is a new method for resolving multiple peaks superimposed on a slowly varying continuum into separate normal (Gaussian) distributions without developing a functional approximation for the continuum. The method is based on fitting the first derivative of the data with the first derivative of the sum of a series of normal distributions. A functional approximation for the continuum is not necessary as long as the first derivative of the continuum is approximately zero (i.e., the continuum varies slowly compared to the normal distributions). © 1993 Wiley‐Liss, Inc.

This article is a US Government work and, as such, is in the public domain in the United States of America.
Recognition and reduction of artifacts from autolysis in paraffin‐embedded tissue using DNA/nuclear protein flow cytometry
Authors: Pollack Alan , Ciancio Gaetano , Terry Nicholas H. A. , Block Norman L.
Published: August 2005

Artifacts from autolysis can be a problem in retrospective flow‐cytometric analyses of DNA content in paraffin‐embedded tissues. Autolyzed tissue from rat liver, human liver, and rat spleen were stained for DNA and nuclear protein to determine if this technique would be useful in identifying partially degraded cells. After the tissue was deparaffinized and rehydrated, the nuclei were isolated using 0.5% pepsin. Propidium iodide (PI) and fluorescein isothiocyanate (FITC) were used to stain DNA and nuclear protein. When unfixed rat liver tissue was allowed to undergo autolysis at 4°C for 24–48 h before fixation, there was a progressive broadening of the G1 and G2M DNA peaks and a slight increase in the average DNA contents of these peaks. Nuclei that stained more intensely with PI also stained more intensely with FITC. Similar results were obtained using human liver and rat spleen. Sometimes the increased PI staining resulted in a false aneuploid peak. The distinctive skewing of the DNA/nuclear protein histograms from autolysis was reduced by increasing the incubation of the tissue in 0.5% pepsin from 0.5 h to 1.5 h during the nuclei‐isolation step. The DNA/nuclear protein method provides a means for identifying artifacts from autolysis, whereas the extended pepsin treatment provides a means for reducing these artifacts. © 1993 Wiley‐Liss, Inc.
Fluorescence instability of propidium iodide‐labelled microspheres in (para)formaldehyde
Authors: Rohr Edgar L. , Kaffenberger Walter
Published: August 2005

An instability of the fluorescence of propidium iodide (PI)‐labelled microspheres was observed when the beads were used as an internal fluorescence standard in cell samples suspended in paraformaldehyde (pFA) for flow cytometric phenotyping. Flow cytometry of fluorochrome‐labelled microspheres as well as spectrofluorometry of PI‐fluorochrome solutions in PBS‐buffered 1% pFA or formalin (FA) revealed strong increases of PI fluorescence starting immediately after the addition to the fixatives. We propose a chemical reaction which leads to two additional conjugated double bonds in the modified fluorochrome causing the increased fluorescence emission. Therefore, PI‐labelled microspheres should not be applied as internal fluorescence references in aldehyde‐containing cell suspensions for flow cytometric analysis, except when the time interval between the addition of beads and the measurement can be kept constant or if it lasts longer than 1 h. © 1993 Wiley‐Liss, Inc.
Equilibrium binding of dapi and 7‐aminoactinomycin D to chromatin of cultured cells
Authors: Rundquist Ingemar
Published: August 2005

The binding of 4′,6‐diamidino‐2‐phenylindole (DAPI) and 7‐aminoactiomycin D (7‐AAMD) to chromatin of intact cultured fibroblast nuclei was studied by cytofluorometry. Staining was performed at equilibrium at varying dye concentrations. By using large volumes of dye solutions, the free dye concentration became approximately equal to total dye concentration, and the estimation of affinity and number of binding sites was performed by Scatchard analysis. Since all Scatchard plots showed a definite curvature, indicating more than one class of binding sites, a two component regression was performed, although only the highest affinity binding sites were determined. The observed affinity constants did not change substantially between different preparational procedures for any of the dyes. The number of DAPI binding sites increased about 30% after detergent extraction. No such increase was observed for 7‐AAMD. Fixation in formaldehyde, instead of ethanol, after detergent extraction had no effect on DAPI binding, but decreased the number of 7‐AAMD binding sites with about 50%. Extraction of basic proteins with HC1 resulted in about 100% increase of the number of binding sites for both dyes. It should be feasible to use this technique as an assay for the identification of proteins or other nuclear components important for the maintenance of chromatin structure. © 1993 Wiley‐Liss, Inc.
Detection of 5‐bromo‐2‐deoxyuridine (BrdUrd) incorporation with monoclonal anti‐brdurd antibody after deoxyribonuclease treatment
Authors: Takagi Shuji , McFadden Marcia L. , Humphreys Robert E. , Woda Bruce A. , Sairenji Takeshi
Published: August 2005

We studied the effects of deoxyribonu‐cleases on the detection of 5‐bromo‐2‐deoxyuridine (BrdUrd) by anti‐BrdUrd monoclonal antibodies (mAbs). After DNase I treatment, BrdUrd was detected in cells fixed on slides with the anti‐BrdUrd mAbs, B44 and BMC9318. The level of detection related to the degree of DNA digestion. DNA digestion of 25–75% resulted in levels of staining comparable to control preparations in which DNA was denatured by heating with formamide. Staining with the mAbs of DNase I‐treated cells was abolished with S1 nuclease, a single‐stranded DNA‐specific nuclease. When exonuclease III was used after DNase I treatment, the staining intensity of cells fixed on slides increased, and BrdUrd could be detected in suspended cells by flow cytometry. Since this enzymatic method leading to the detection of BrdUrd does not involve cell loss, or destruction of either cellular morphology or epitope reactivity, as occurs with traditional DNA denaturation procedures, it is useful for kinetic studies of phenotypically mixed populations. Furthermore, staining with anti‐BrdUrd mAb of cells treated with exonuclease III offers a simple approach to quantitation of apoptotic cells, in which an endogenous endonuclease is activated. © 1993 Wiley‐Liss, Inc.
Statistical methods in interphase cytogenetics: An experimental approach
Authors: Kibbelaar R. E. , Kok F. , Dreef E. J. , Kleiverda J. K. , Cornelisse C. J. , Raap A. K. , Kluin Ph. M.
Published: August 2005

In situ hybridization (ISH) techniques on interphase cells, or interphase cytogenetics, have powerful potential clinical and biological applications, such as detection of minimal residual disease, early relapse, and the study of clonal evolution and expansion in neoplasia. Much attention has been paid to issues related to ISH data acquisition, i.e., the numbers, colors, intensities, and spatial relationships of hybridization signals. The methodology concerning data analysis, which is of prime importance for clinical applications, however, is less well investigated. We have studied the latter for the detection of small monosomic and trisomic cell populations using various mixtures of human female and male cells. With a chromosome X specific probe, the male cells simulated monosomic subpopulations of 0, 1, 5, 10, 50, 90, 95, 99, and 100%. Analogously, when a (7 + Y) specific probe combination was used, containing a mixture of chromosome No. 7 and Y‐specific DNA, the male cells simulated trisomic cell populations. Probes specific for chromosomes Nos. 1, 7, 8, and 9 were used for estimation of ISH artifacts. Three statistical tests, the Kolmogorov‐Smirnov test, the multiple‐proportion test, and the z'‐max test, were applied to the empirical data using the control data as a reference for ISH artifacts. The Kolmogorov‐Smirnov test was found to be inferior for discrimination of small monosomic or trisomic cell populations. The other two tests showed that when 400 cells were evaluated, and using selected control probes, monosomy X could be detected at a frequency of 5% aberrant cells, and trisomy 7 + Y at a frequency of 1%. A simple‐proportion test was developed for illustration of the quantitative constraints of interphase cytogenetics. For detection of 1% aberrant cells and given an ISH artifact rate of 10%, 10,173 cells must be evaluated. These results indicate that automated ISH evaluation is imperative for clinical and biological applications. © 1993 Wiley‐Liss, Inc.
Phenotype analysis of cycling and postcycling thymocytes: Evaluation of detection methods for BrdUrd and surface proteins
Authors: Penit Claude , Vasseur Florence
Published: August 2005

We present a comparison of two different methods for simultaneous detection of bromodeoxyuridine and cell surface markers. Both methods use enzymatic generation of single‐etrand DNA with nuclease. The biological system used is the murine thymus, in which in vivo DNA synthetizing cells were labeled by injection of BrdUrd and analyzed at different time points after the nucleoside pulse. The surface proteins detected were CD4 and CD8 differentiation markers and the T‐Tell receptor. Extraction of DNA‐associated proteins with 0.1N HC1 and detergent is necessary for the action of EcoR1 and Exonuclease III, but this treatment destroys phycocyanins and induces cell aggregation, as shown using the doubletdiscrimination module. For DNAse I action, cells could be treated with paraformaldehyde and a low concentration of Tween 20, and this treatment was adequate for surface staining preservation (even writh phycocyanins) and BrdUrd detection. Both methods were adequate for cell cycle studies, but only 7‐aminoactinomycin D could be used as total DNA dye after DNAse action, and good results needed long (48–72 h) incubation in the fixative‐eetergent mixture. The DNAse I method now allows three‐color staining (two surface markers and BrdUrd), analyzed in a one laser‐cytometer for the study of the phenotype of cycling cells, and of their progeny, in vivo and in cell cultures. It also allows the quantitative analysis of cell surface receptor densities in conditions similar to fresh cells. © 1993 Wiley‐Liss, Inc.
Analysis of lymphocyte activation and proliferation by video microscopy and digital imaging
Authors: Teague T. Kent , Munn Lance , Zygourakis Kyriacos , McIntyre Bradley W.
Published: August 2005

Video microscopy and digital imaging were used as a noninvasive method to quantitatively analyze lymphocyte activation and proliferation. This method takes advantage of the fact that upon activation lymphocytes blast and become significantly larger before proliferating. The mean cell sizes of T lymphocytes in an activation kinetics assay were measured by digital image analysis and compared to [3H]‐thymidine incorporation of cells under the same treatment. An increase in cell size was observed before [3H]‐thymidine incorporation; therefore the digital imaging assay is more sensitive in determining the earliest time‐point of activation. Also, the digital imaging assay was comparable to the [3H]‐thymidine incorporation assay in providing information about the extent and rates of T lymphocyte proliferation. Cellular DNA was stained with propidium iodide to show that the larger blasting cells in the population of activated T lymphocytes were indeed the cells that accounted for the increase in DNA synthesis and thus an increase in cell size can be correlated with activation. © 1993 Wiley‐Liss, Inc.
Flow cytometric DNA analysis of wheat‐rye addition lines
Authors: Bashir A. , Auger J. A. , Rayburn A. Lane
Published: August 2005

The nuclear DNA content of the 7 wheat‐rye chromosome addition lines were determined by flow cytometry. The nuclear DNA content of each line was compared to the original wheat parental line. From this information, the amount of DNA per rye chromosome was calculated. The DNA per chromosome ranged from 0.8 picograms (pg) in chromosome 3R to 1.4 pg in chromosome 7R. Flow cytometry was proven to be a rapid and reliable technique for the evaluation of aneuploid plant populations. Flow cytometry has the potential to allow for more rapid screening of aneuploid and euploid cytological stocks than conventional cytogenetic techniques. © 1993 Wiley‐Liss, Inc.
Germinal center analysis with the tools of mathematical morphology on graphs
Authors: Raymond Eric , Raphael Martine , Grimaud Michel , Vincent Luc , Binet Jacques Louis , Meyer Fernand
Published: August 2005

Only few studies devoted to quantitative analysis of tissue architecture have been performed. The analysis of neighborhood relationships between cells, using graphs and mathematical morphology (MM), constitutes one approach. We propose to analyse quantitatively the architecture of a tissue with the tools of MM on graphs. The use of graphs seems best suited to take into account the neighborhood relationships between cells, independently from their mutual distances: two cells are considered as neighbors if no interfering cell is placed in a given sense between them. Such neighboring cells are linked by an arc in a graph. On such a graph, all tools of MM may be applied. We investigate two of them in the present work: (1) the distance transforms permits to analyse the repartition of a cellular population A relative to population B, (2) the size distribution permits to analyse the tendancy of a cell population to form clusters. We have applied this method using Gabriel's graph, derived from the Voronoï diagram, to determine the “zone of influence” of a cell.

We have analysed the neighborhood relationships between cells in germinal centers (GC) from lymph rodes. Twelve hyperplastic GCs from follicular hyperplasia (FH) and 5 neoplastic GCs from small cleaved cell follicular lymphoma (SCCFL) have been studied; 2 m̈m hematoxylin‐eosin plastic embedded sections have been analysed. Cell nuclei have been identified manually by the observer by giving a numerical and image color code. Gabriel's graphs have been constructed with all centrofollicular cells. Subgraphs with only lymphoïd cells, large cells or small cleaved cells have also been studied.

The application of MM transformations on the graphs using software package Morphograph has allowed a quantitative description of cell distribution in the tissue. The distances of centrofollicular lymphoïd cells from the periphery of the GCs (mantle zone), from nonlymphoïd cells as macrophages located within the GCs and between large lymphoïd cells and small lymphoïd cells have been determined in FH and SCCFL. Using iterative closings and openings, we have detected aggregates of small and large cells and characterized their size and distribution in the GCs. The application of this method on lymph node biopsy has allowed a quantitative description and comparison of GCs in different pathological conditions. © 1993 Wiley‐Liss, Inc.
Flow cytometric analysis of the expression of PCNA during the cell cycle in hela cells and effects of the inhibition of DNA synthesis on it
Authors: Sasaki Kohsuke , Kurose Akira , Ishida Yoji
Published: August 2005

Flow cytometric bivariate DNA/PCNA analysis was performed to investigate the expression of PCNA during the cell cycle and the implication in DNA replication in HeLa cells, using a monoclonal antibody (PC10) to PCNA. The expression of PCNA was evident in almost all cells growing exponentially, when cells were fixed in methanol. The total amount of PCNA altered a little during the cell cycle. However, the treatment with Triton X‐100 extracted 80–89% of total PCNA from the cells, resulting in the dramatic change of bivariate DNA/PCNA distribution pattern. PCNA was completely removed from nuclei in both G1 and G2 phases by the detergent treatment, whereas a certain amount of PCNA remained in S phase nuclei. After the treatment of cells with Triton X‐100, PCNA was detected exclusively in S phase cells. The bivariate DNA/PCNA distribution pattern in cells treated with Triton X‐100 was strikingly so similar to the DNA/BrdUrd distribution pattern that it was unable to differentiate one from the other. It is concluded that the detergent treatment of cells allows the rapid analysis of the cell cycle.

The inhibition of DNA synthesis with 10 mM hydroxyurea elevated cellular PCNA content mainly due to the increase in the fraction of the detergent extractable PCNA. It was apparent, however, that in cells incubated with Triton X‐100, the pattern of the bivariate DNA/PCNA distribution was not basically different from that in cells without HU treatment. The level of PCNA bound to nuclear structures (PCNA not extracted with detergent) increased in cells arrested at the G1/S boundary with the time of hydroxyurea treatment. Eventually, it became difficult to differentiate between cells in the G1/S boundary and the early S phase on the basis of DNA and PCNA contents. However, the increase in bound PCNA was minimal in S phase cells. The observation suggests that PCNA is bound to the potential initiation sites of DNA replication together with DNA replication associated enzymes in advance of the commencement of DNA replication and that it is never associated with late replicating DNA without the duplication of early replicating DNA. © 1993 Wiley‐Liss, Inc.
Development and optimization of tissue preparative methodology for DNA content analysis of soft tissue neoplasms
Authors: Zalupski Mark M. , Ryan James R. , Ensley John F. , Maciorowski Zosia , Pietraszkiewicz Haline , Hussein Mohamed E. , Kukuruga Mark , Sundareson Alistair S. , Baker Laurence H.
Published: August 2005

Data regarding DNA content parameters in soft tissue sarcoma is limited. Development and optimization of tissue specific preparative techniques for DNA flow cytometry was undertaken prior to routine DNA content analysis of soft tissue neoplasms; 154 soft tissue tumors were studied. Dissociation dependent differences in cellular yields, viabilities, maintenance of DNA aneuploid populations, coefficients of variation, and DNA index supported the need for these developmental studies. Fifty‐six of eighty‐nine patients had DNA aneuploid soft tissue sarcomas. A relationship between DNA aneuploidy and grade was seen in this series with 38% with low grade, 59% with moderate grade, and 69% with high grade tumors demonstrating DNA aneuploid populations (P < 0.005). The mean S‐phase fraction for DNA diploid and aneuploid sarcomas was 7.2% and 13.3%, respectively (P ≤ 0.001). When classified by histologic grade of the primary tumor, a greater percentage of metastatic lesions were DNA aneuploid (4 of 7 grade 2 lesions, and 15 of 16 grade 3 lesions). Decreases in cellular yields and rate of DNA aneuploidy were observed in a subgroup of patients with localized high grade sarcoma treated preoperatively, as compared to patients treated with initial surgery. Prospective correlation of DNA content parameters to prognosis and response to cytotoxic therapy are now possible and are ongoing. © 1993 Wiley‐Liss, Inc.
Improved method for release of cell nuclei from paraffin‐embedded cell material of squamous cell carcinomas
Authors: Wang Nainig , Pan Yi , Heiden Thomas , Tribukait Bernhard
Published: August 2005

An improved method of releasing cell nuclei from paraffin‐embedded highly keratinized squamous cell carcinomas by pretreatment with 85% formic acid‐0.3% H2O2 followed by enzymatic treatment with subtilisin Carlsberg is described. After DAPI staining the resulting suspensions of cell nuclei were analyzed by DNA flow cytometry, in addition to microscopy. The total yield of released cell nuclei was improved and the proportion of tumor cell nuclei in the suspensions increased from 12% to 53% using this method compared to the conventional preparation technique. Cytoplasmic residue disappeared nearly completely. Thus, the finding of false aneuploid cell populations representing diploid cells with autofluorescent cytoplasm could be avoided. In addition, even small true aneuploid cell populations could be detected due to the increased proportion of released tumor cell nuclei and the lower proportion of background in the >2C region. © 1993 Wiley‐Liss, Inc.
Prevention of nutritionally induced diabetes by rosiglitazone in the gerbil Psammomys obesus
Authors: Hefetz Simona , Ziv Ehud , Jörns Anne , Lenzen Sigurd , Shafrir Eleazar
Published: August 2005

Background

Psammomys obesus is a desert gerbil developing hyperglycaemia, hyperinsulinaemia and insulin resistance when placed for 2 weeks on a high‐energy (HE) diet. The mechanism underlying the antidiabetic effect of rosiglitazone (RG) treatment (20 mg/kg per day for 2 weeks) was studied.

Methods

The antidiabetogenic effect of RG treatment on serum insulin and metabolic parameters in serum and target tissues of insulin action was investigated in vivo and compared with the pancreatic beta cell protective effects of RG.

Results

Almost all RG‐treated animals remained normoglycaemic compared to controls, but, at the same time, they were hyperinsulinaemic. RG had no effect on serum free fatty acid and serum and muscle triglyceride concentrations and did not appreciably affect body weight and fat depots. RG prevented a HE diet‐induced reduction of GLUT 4 glucose transporter content in epididymal adipose tissue, but not in gastrocnemius muscle. The normoglycaemic effect was not associated with a suppression of liver PEPCK activity. Muscle PKCε expression, known to be elevated in diabetic Psammomys and to inhibit insulin signalling, was only marginally decreased. However, RG treatment prevented the marked decrease in insulin immunostaining as well as the vacuolization of the beta cells and accelerated beta cell proliferation.

Conclusions

These data indicate that the skeletal muscle is not the primary target of RG action, whereas the preservation of the insulin secretory capacity and the prevention of degenerative beta cell vacuolization in spite of persisting insulin resistance appear to be the basis for the anti‐hyperglycaemic effect of RG in Psammomys. Copyright © 2005 John Wiley & Sons, Ltd.
Lithium tert‐Butyl[phenyl(2,2,6,6‐tetramethylpiperidino)boryl]amide: A Versatile Reagent
Authors: Braun Ulrike , Habereder Tassilo , Nöth Heinrich
Published: August 2005

Figure 1 on page 3632 of the original article1 is actually a copy of Figure 2; the correct Figure 1 is shown below.
Enhanced amperometric detector for local anesthetics in liquid chromatography with metal‐oxide dispersed glassy carbon electrodes
Authors: Zhou Jianxun , Zhang Lijing , Wang Erkang
Published: August 2005

Chemically modified electrodes (CMEs) prepared by the dispersion of metal oxide particles on a glassy carbon (GC) substrate greatly enhance the voltammetric response and amperometric detection of local anesthetics following liquid chromatography (LC). The enhancement is more pronounced with the GC electrodes dispersed by the metal oxides of higher oxidation states (+3, +4) and for the species exhibiting relatively slow electrode kinetics under given conditions. With an applied potential of 1.2 V (vs. SCE), LC amperometric detection of the analytes at the α‐alumina modified GC surface gives detection limits 2–5 times lower than those obtained at the bare electrode. The metal oxide‐dispersed electrodes display significant improvement in sensitivity, and selectivity and indicate excellent preparation reproducibility and performance stability.
Application of partial least‐squares regression to the suitability of multicomponent polarographic determination of organochlorine pesticides in emulsified medium
Authors: Reviejo A Julio , Buyo Federico J. , Pingarrón José M. , Peral José L.
Published: August 2005

Partial least‐squares regression (PLSR) has been used for treatment of the polarographic data obtained from mixtures of dieldrin, heptachlor, endosulfan, and endosulfan sulfate in an emulsified medium formed with ethyl acetate and a mixture of Triton X‐405 and Hyamine 2389 as emulsifying agent. Pesticide concentrations were within the range of 1.0 × 10−6 to 9.0 × 10−6 mol L−1. The size of the calibration set was 35 samples by nine current measurements at different potentials. The overall „poorness”︁ or „richness”︁ of a sample can be predicted by plotting the samples in the space of the first two latent variables of the intensities matrix. The model was improved by constructing a new calibration set composed of samples containing only three and four components. In order to distinguish among the different types of samples (binary, ternary, etc.) a linear cutoff function was defined by dividing the sampling space into two calibration zones. Another assignment criterion for both calibration sets was the trunked Mahalanobis distances. External validation of the calibration sets was accomplished with seven new „unknown”︁ samples. All of these samples were properly assigned using the cutoff linear function, and prediction of the pesticides concentration in each sample was possible with acceptable errors.
Determination of heavy metals in concentrated refined sugar and raw syrups with differential pulse polarography and anodic stripping voltammetry
Authors: Khoulif Zidane , Jambon Claude , Chatelut Michelle , Vittori Olivier
Published: August 2005

The behavior of copper, lead, cadmium, and zinc is investigated in concentrated solutions of refined beetroots sugar. The diffusion coefficients are found to decrease less than predicted by the Walden's rule, so trace determinations down to 10−7 M were measured by differential pulse polarography (DPP). Using anodic stripping voltammetry at a mercury film electrode, it was found that the refined sugar of alimentary grade contained: 57 (Cu), 34 (Zn), 1 (Cd), and 6 (Pb) μg/kg of dry sugar. For industrial syrups, resulting from a treatment by lime and water vapor of the raw material, some problems were encountered. Determination of copper and zinc was impossible, while that of lead and cadmium was possible after one liter of diluted syrup, at about 250 g l−1, had been treated with 20 g of activated carbon to absorb the major part of organic impurities. The lead and cadmium contents were found to be 20 (Pb) and 3.5 (Cd) μg kg−1.
A continuous time Markov chain model for a plantation‐nursery system
Authors: Gani J. , Stals L.
Published: August 2005

This article examines a continuous time Markov chain model for a plantation‐nursery system in which diseased plantation trees are replaced at a daily rate λ by nursery seedlings. There is a random infection rate α caused by insects, and the disease is also spread directly between the N plantation trees at the rate β, starting with a diseased trees at time t = 0; in addition, some replacement seedlings prove to be infected with probability 0 < p. < 1. We find a formal solution to the system in terms of the Laplace transforms $\hat p_j$, j = 0,…, N, of the probabilities pj(t) of j infected plantation trees at time t. A very simple example for N = 2, a = 1 is used to illustrate the method. We then consider numerically the effect of the parameters λ, α, and β on the system, and for small t study the influence of the initial number a of infected trees on the expected number of such trees at time t ≤ 365. As t → ∞, stationarity is achieved, irrespective of the initial value a. Copyright © 2005 John Wiley & Sons, Ltd.
Numerical simulation of the miscible displacement of radionuclides in a heterogeneous porous medium
Authors: Bruneau C.‐H. , Marpeau F. , Saad M.
Published: August 2005

The aim of this paper is to model and simulate the displacement of radioactive elements in a saturated heterogeneous porous medium. New schemes are proposed to solve accurately the convection–diffusion–reaction equations including nonlinear terms in the time derivative. Numerical tests show the stability and robustness of these schemes through strong heterogeneities of the medium. Finally the COUPLEX1 benchmark concerning the far field simulation of a polluted flow by a leak of a nuclear waste disposal is performed and compared with the results available in the literature. Copyright © 2005 John Wiley & Sons, Ltd.
A combined vortex and panel method for numerical simulations of viscous flows: a comparative study of a vortex particle method and a finite volume method
Authors: Kim Kwang‐Soo , Lee Seung‐Jae , Suh Jung‐Chun
Published: August 2005

This paper describes and compares two vorticity‐based integral approaches for the solution of the incompressible Navier–Stokes equations. Either a Lagrangian vortex particle method or an Eulerian finite volume scheme is implemented to solve the vorticity transport equation with a vorticity boundary condition. The Biot–Savart integral is used to compute the velocity field from a vorticity distribution over a fluid domain. The vorticity boundary condition is improved by the use of an iteration scheme connected with the well‐established panel method. In the early stages of development of flows around an impulsively started circular cylinder, and past an impulsively started foil with varying angles of attack, the computational results obtained by the Lagrangian vortex method are compared with those obtained by the Eulerian finite volume method. The comparison is performed separately for the pressure fields as well. The results obtained by the two methods are in good agreement, and give a better understanding of the vorticity‐based methods. Copyright © 2005 John Wiley & Sons, Ltd.