bluuebunny/crossref_metadata_2025_split huggingface dataset:
Statistical confirmation that immunofluorescent detection of DNA repair in human fibroblasts by measurement of bromodeoxyuridine incorporation is stoichiometric and sensitive
Authors: Selden Jules R. , Dolbeare Frank , Clair James H. , Nichols Warren W. , Miller Judith E. , Kleemeyer Katherine M. , Hyland Robert J. , DeLuca John G. 
Published: August 2005

AbstractDiploid human fibroblasts (IMR‐90 cells), grown to confluency and growth‐arrested by serum starvation, were irradiated with a variety of doses of UV light (0.025–40 J/m2) or incubated with broad dose ranges of four direct‐acting mutagens [ethyl methanesulfonate (EMS), ICR‐170, methyl methanesulfonate (MMS), and 4‐nitroquinoline oxide (4‐NQO)] and pulsed with a thymidine analog, 5‐bromodeoxyuridine (BrdUrd) to detect evidence of DNA repair. These cells and appropriate controls were immunochemically stained and subjected to flow cytometric analysis to quantify BrdUrd incorporation into DNA and simultaneously measure cellular DNA content. Since the maximal quantity of BrdUrd incorporated with repairing cells is profoundly less than the amount incorporated during replicative synthesis and flow cytometric analysis collects information on a cell‐to‐cell basis, data collection using linear histograms succeeded both in revealing repairing cellular populations and eliminating replicative cells from the analysis. Technical modifications necessary to achieve stoichiometry with UV‐irradiated IMR‐90 fibroblasts included a 48h repair (and pulse) period, followed by denaturing cellular DNA for 15 min at 90°C. The limit of detection was equal to or below the lowest dose investigated (0.025 J/m2). DNA repair was also detected with cultures incubated with low doses of all chemicals and pulsed with BrdUrd for a 24 h period. The limits of detection were near or below 500 m̈M EMS, 5 m̈M MMS, 0.25 m̈M 4‐NQO, and 0.1 m̈M ICR‐170. © 1993 Wiley‐Liss, Inc.
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

AbstractA 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

AbstractDespite 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

AbstractPreviously 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

AbstractA 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

AbstractWe 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

AbstractWe 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

AbstractFlow 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

AbstractAn 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

AbstractDNA 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

AbstractFlow 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

AbstractThe 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.
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

AbstractWhen 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

AbstractDerivative 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

AbstractArtifacts 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

AbstractAn 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.