bluuebunny/crossref_metadata_2025_split huggingface dataset:
Cell kinetic effects of incorporated 3H‐thymidine on proliferating human lymphocytes: Flow cytometric analysis using the DNA/nuclear protein method
Authors: Pollack A. , Moulis H. , Greenstein D. B. , Block N. L. , Irvin G. L. 
Published: August 2005

AbstractPhytohemagglutinin‐stimulated human peripheral blood lymphocytes incorporating high concentrations of 3H‐thymidine accumulate in G2 and show a consequent reduction in the number of cells entering M (division delay). The simultaneous flow cytometric analysis of DNA content (propidium iodide fluorescence) and nuclear protein content (fluorescein isothiocyanate fluorescence) allows for the accurate quantitation of these events; G2 and M are separated in the bivariate distributions. A good correlation was observed between mitotic indices, quantitated by manually counting mitotic cells, and integration of the M area in DNA/nuclear protein histograms. Moreover, significant differences in G2 nuclear protein levels were found between untreated and 3H‐thymidine‐treated lymphocytes. In order to characterize this effect, G2 was empirically divided into low nuclear protein (G2A) and high nuclear protein (G2B) compartments. 3H‐thymidine caused an initial accumulation of lymphocytes in G2A, followed within 3–6 h by a gradual movement of some cells into G2B, with a subsequent accumulation of cells in G2B. The results suggest that the distribution of cells in G2 (G2A and G2B), the average nuclear protein content of G2B cells, and the proportion of cells in M are parameters that when used in combination provide a unique description of radiobiological effects.
Anthracycline resistance and consequences of the in situ–in vitro transfer
Authors: Šonka J. , Stöhr M. , Vogt‐Schaden M. , Volm M. 
Published: August 2005

AbstractAdriamycin‐resistant and normal cells of the sarcoma 180 of the mouse undergo qualitatively different deflections from the in situ state when prepared for an experiment. Resistant cells perform a fast reactive decline in the proliferative activity. They are capable of quiescence as defined by the time needed for the induction of the proliferation. Sensitive cells seem to be unable to quiesce and are only slowed down. These facts must be taken into account in interpretation of similar results. Differences in experiments need not necessarily imply differences in situ. Such in vitro appearing differences between sensitive and adriamycin‐resistant cells of the murine sarcoma 180 include the retention of the mitochondria‐specific stain rhodamine 123 and the uptake of anthracyclines, both being reduced in resistant cells.After labeling sensitive cells with thymidine in vivo and sorting them according to their rhodamine 123‐derived fluorescence, the label was only found in the major, highly fluorescing fraction. A small low‐fluorescing fraction remained unlabeled. We were able to demonstrate similar results with labeled anthracyclines applied to both the sensitive and the resistant cells in a short period between the removal of the cells from the ascites and the cells sorting. The adriamycin resistance seems to be joined with the ability of the cells to reduce their proliferative activity following changes to unfavorable conditions in vitro. Quiescent cells of the resistant line demonstrate the “anthracyclin pump.” Substances which are known to increase the sensitivity of anthracycline‐resistant cells (TWEEN, verapamil) also shift the cells from low to high rhodamine 123‐fluorescence.
Flow cytometry of human colorectal tumors: Nuclear isolation by detergent technique
Authors: Petersen Steffen E. 
Published: August 2005

AbstractA simple one‐step technique for detergent isolation and DNA staining of nuclei from mouse colon and from human colorectal tumors was investigated. Nuclear yield increased with treatment time, up to 24 h. There were only minor differences when detergent concentrations from 0 to 0.6% were used. The lowest (0.03%) concentration was most effective. No loss of nuclei was effected by cell lysis and no selectivity was observed for isolation of certain cell‐cycle phases or ploidy classes from heterogeneous tumors. The nuclei were stable in the stain‐detergent solution for 24 h, but lymphocytes were sensitive to the possible proteolytic activity of one of the two commercial RNase preparations. Of the total number of parenchymal nuclei in mouse liver, as estimated by a stereological method, approximately 60% were isolated by the procedure (approximately 0.9 × 108 nuclei/g tissue). From mouse colon the average nuclear yield was 1.8 × 108/g, and from human colorectal tumors 0.9 × 108/g (ranges 0.3–1.9 × 108). Microscopic examination of undissolved tissue fragments from the preparation of tumors and mouse colon showed a high selectivity for isolation of epithelial and neoplastic nuclei, leaving the stroma with its nuclei almost intact.
A monoclonal antibody reactive with 5‐bromo‐2‐deoxyuridine that does not require DNA denaturation
Authors: Gonchoroff Nick J. , Greipp Philip R. , Kyle Robert A. , Katzmann Jerry A. 
Published: August 2005

AbstractWe describe a mouse monoclonal antibody (BU‐1) reactive with 5‐bromo‐2‐deoxyuridine (BrdUrd). The antibody is different from previously described BrdUrd monoclonal antibodies in that BU‐1 does not require pretreatment of cells with strong DNA denaturants in order for the antibody to react with BrdUrd incorporated in the DNA. The antibody can be used in immunocytochemical and indirect immunofluorescent assays and can be used to identify cells that have incorporated BrdUrd. Double staining with BU‐1 antibody and propidium iodide has been used to confirm S‐phase measurements with the BU‐1 antibody. Immunocytochemical stains using the BU‐1 antibody do not destroy cell morphology and allow cell identification to be performed simultaneously with S‐phase measurements. Flow Cytometer two‐color fluorescence analysis allows the simultaneous identification of cell surface or cytoplasmic markers and S‐phase quantitation. The BU‐1 antibody should broaden the application of cell kinetic measurements to individual elements of cell populations that are heterogeneous with respect to morphology, surface marker, and other biological features.
Simultaneous analysis of cell surface antigens, bromodeoxyuridine incorporation and DNA content
Authors: Houck David W. , Loken Michael R. 
Published: August 2005

AbstractA method has been developed for correlating the presence of cell surface markers with bromodeoxyuridine (BrdUrd) uptake. We have found that paraformaldehyde fixation will maintain immunofluorescent cell surface staining through acid denaturation that is necessary for anti‐BrdUrd reactivity to single‐stranded DNA. In addition, the forward angle light scattering was maintained, eventhough the cells had been exposed to 2 N HCl and detergent. The protocol was tested on three model systems: mouse thymus, a human cell line (CCRF‐SB), and peripheral blood leukocytes. It was found that there was no specific loss of lymphocyte subsets.
Multivariate analysis and list mode processing of murine hemopoietic subpopulations for cytokinetic studies
Authors: Pallavicini M. G. , Summers L. J. , Giroud F. J. , Dean P. N. , Gray I. W. 
Published: August 2005

AbstractMultivariate analyses and list mode data processing were used to obtain cytokinetic information on flow cytometrically distinct hemopoietic subpopulations. In one application, viable, unfixed hemopoietic subpopulations were discriminated on the basis of cyanine dye fluorescence and orthogonal light scatter; Hoechst dye fluorescence was measured to determine the proliferative status of the subpopulations. In another application, ethanol‐fixed mouse bone marrow cells were triply stained with Hoechst dye, rhodamine‐conjugated wheat germ agglutinin (WGA), and a fluorescein‐labeled monoclonal antibody against bromodeoxyuridine. In both of these studies, flow cytometric data for all three variables were acquired in list mode fashion, stored on magnetic tape, and processed by list mode software on a computer based multivariable pulse‐height analyzer. In the first application, subpopulations distinguished by cyanine dye intensity and light scatter appeared to be more related to cell lineage and cell size than proliferative status. In the second application, WGA affinity discriminated two subpopulations in mouse bone marrow S‐phase cells in each subpopulation that actively incorporated bromodeoxyuridine (BrdUrd). List mode data processing obviates the need for routine electronic sorting of cells and thus facilitates characterization of discriminated subpopulations. In this regard, it is particularly useful for the study of flow cytometrically distinct, low frequency subpopulations.
Mapping of the pattern of DNA replication in polytene chromosomes from Chironomus thummi using monoclonal anti‐bromodeoxyuridine antibodies
Authors: Allison Linda , Arndt‐Jovin Donna J. , Gratzner Howard , Ternynck Thérèse , Robert‐Nicoud Michel 
Published: August 2005

AbstractWe present results from a nonautoradiographic study of DNA replication in polytene chromosomes from dipteran larvae. Monoclonal antibodies with specificity for 5‐bromodeoxyuridine (BrdUrd) were used to localize by indirect immunofluorescence the sites of BrdUrd incorporation and to follow the dynamics of DNA synthesis in salivary gland cells of 4th instar Chironomus thummi larvae. This technique presents numerous advantages over autoradiographic procedures and allows mapping of DNA synthesis patterns at the level of resolution of one chromosomal band. Several replication patterns were observed, classified according to characteristic features, and tentatively assigned to specific periods of the S‐phase. In early S‐phase, DNA synthesis is first detectable in puffs and inter‐bands, later in bands. Most chromosomalbands appear to initiate DNA synthesis synchronously; however, in bands within centromeric and heterochromatic regions the start of synthesis is delayed. At mid S‐phase, all the bands show uniform staining. Subsequent staining patterns are increasingly differential with the bands displaying characteristic fluorescence intensities. As replication progresses through the late S‐phase period, the chromosomes show a decreasing number of fluorescent bands. The last bands to terminate replication are located in centromeric and heterochromatic DNA‐rich regions and a few bands of low DNA content in region IIAa‐c.
The use of FITC‐conjugated gated monoclonal antibodies for determination of S‐phase cells with fluorescence microscopy
Authors: Gunduz Nurten 
Published: August 2005

AbstractA method is modified to determine the DNA synthesizing cells in primary human breast tumors and cells with idiopathic thrombocytopenic purpura (ITP) with FITC‐conjugated monoclonal antibody against bromodeoxyuridine (FITC‐M‐anti‐BrdUrd) and fluorescence microscopy. The DNA synthesizing cells were also determined from a portion of the same tissues by classical tritiated thymidine labeling (3HdThd) and autoradiography. The results from bromodeoxyuridine labeling index (BrdUrd‐LI) and tritiated thymidine labeling index (3HdThd‐LI) obtained from the same tissues were compared. The mean BrdUrd‐LI for breast tumor was 5.4 ± 1.0% and the mean 3HdThd‐LI was 5.5 ± 1.1%. Similarly, the labeling indexes obtained from mononuclear leukocytes of healthy donors had means of 0.5 ± 0.1% and 0.6 ± 0.1% for BrdUrd‐LI and 3HdThd‐LI, respectively. The change in the proliferation rate of mononuclear leukocyte population in the samples obtained from patients with ITP could be observed by both methods. The mean BrdUrd‐LI of mononuclear leukocytes for this hematological disorder was 5.4 ± 0.9% and the mean 3HdThd‐LI was 6.1 ± 0.8%. These results suggest that this relatively simple technique offers an alternative method for determining the DNA synthesizing cells in a given cell population.
Direct and indirect flow cytomyetric enumeration of 6‐thioguanine‐resistant human peripheral blood lymphocytes
Authors: Matsson P. , Amnéus H. , Djupsjöbacka M. , Eriksson B. , Eriksson L. , Fellner‐Feldegg H. , Zetterberg G. 
Published: August 2005

AbstractMethods based on flow cytometry and sorting, auto radiography, and cloning were used to evaluate the potential for the enumeration of 6‐thioguanine‐resistant human peripheral blood lymphocytes assumed to be deficient with respect to the enzyme hypoxanthineguanine‐phosphoribosyl‐transferase. Flow cytometric sorting of proliferating cells in the late S‐and the G2‐stages by means of DNA content, as measured by propidium iodide fluorescence, enabled an enrichment of variant cells to about 99%. The main source of false events was contaminating doublets of G0/G1 cells appearing in the sorting region. Doublet discrimination measured as the difference between pulse height and area (Ortho‐50) accomplished no further improvement. A combination of propidium iodide fluorescence and bromodeoxyuridine incorporation, measured by fluorescent anti‐bromodeoxyuridine‐DNA antibodies, allowed flow cytometric enrichment to about 99.99% of variant cells.By sorting of 3H‐thymidine‐labeled cell nuclei from the late S‐and the G2‐phases and subsequent autoradiographic evaluation, partly resistant variants could be discriminated; variant frequencies of the same magnitude as for the cell cloning methods were obtained.
Detection of cytosine arabinoside resistant cells at low frequency using the bromodeoxyuridine/DNA assay
Authors: Waldman Frederic M. , Dolbeare Frank , Gray Joe W. 
Published: August 2005

AbstractThis report describes a rapid and sensitive procedure for detection of cytosine arabinoside‐ (Ara‐C) resistant mouse leukemia cells (L1210) in a predominantly Ara‐C‐sensitive population. L1210 cell lines sensitive or resistant to Ara‐C were grown and treated with Ara‐Cin vitroorin vivo. Ara‐C‐resistant cells were detected as those cells with S‐phase DNA content retaining the ability to incorporate bromodeoxyuridine (BrdUrd) after treatment with Ara‐C. The BrdUrd incorporation ability of the S‐phase cells was assessed by simultaneous flow cytometric measurement of cellular DNA content and amount of incorporated BrdUrd. The proportion of Ara‐C‐resistant cells was accurately estimated at frequencies approaching 10−3.
Cytopress: Automated slide preparation of cytologic material from suspension
Authors: Oud Peter S. , Haag Ditty J. , Zahniser David J. , Ramaekers Frans C. S. , Huysmans Anita C. L. M. , Veldhizen José A. M. , Verheyen René H. M. , Verrijp Kiek , Broers Jos L. V. , Herman Chester J. , Vooijs G. Peter 
Published: August 2005

AbstractThis paper describes a new automated system to prepare slides of cytological material from suspension. The system collects material on a filter tape by filtration and transfers it to glass slides by means of pressure‐fixation. Using cervical cells as a model, results show that a well‐defined cell number is evenly deposited over a standardized area, while a small number of cells is retained on the tape and a negligible number lost in the filtrate. Contamination is very small. Application of the system to other cytological material (fine needle aspirations, monolayer and cell suspension cultures, agar cultures, and isolated nuclei) is shown. In general, more than one slide can be made from one sample. Several histological staining procedures as well as immunofluorescence labeling protocols can be applied to the preparations obtained in this way. This system thus introduces a method that will standardize specimen preparation, is quick, saves operator time, and can be used for both diagnostic and research applications.
Changes in accessibility of DNA to various fluorochromes during spermatogenesis
Authors: Evenson D. , Darzynkiewicz Z. , Jost L. , Janca F. , Ballachey B. 
Published: August 2005

AbstractAccessibility of mouse testicular and vas deferens (vas) sperm cell DNA to acridine orange, propidium iodide, ellipticine, Hoechst 33342, mithramycin, chromomycin A3, 4′6‐dimidino‐2‐phenylindole (DAPI), and 7‐amino‐actinomycin D (7‐amino‐AMD) was determined by flow cytometry. Permealized cells were either stained directly or after pretreatment with 0.06 N HCL. For histone‐contanining tetraploid, diploid, and round spermatid cells, HCI extraction of nuclear proteins caused an approximately sixfold increase of 7‐amino‐AMD stainability but had no signicant effect on DAPI stainability. For these same cell types, the stainability with other intercalating (acridine orange, propidium iodide, ellipticine) and externally binding (Hoechst 33342, mithramycin, chromomycin A3) dyes was increased by 1.6‐ to 4.0‐fold after HCI treatment. In sharp contrast, HCI treatment of vas sperm did not increase the staining level of 7‐amino‐AMD, DAPI, or propidium iodide but did increase the staining level for the other intercalating dyes (1.3‐ to 1.5‐fold) and external dyes (1.3‐ to 1.9‐fold). Elongated spermatids that contain a mixture of protein types including histones, transition proteins, and protamines demonstrated the greatest variability of staining with respect to type of stain and effect of acid extraction of proteins. In general, for nearly all dyes, the round spermatids had an increased level and tetraploid cells had a decreased level of stainability relative to the same unit DNA content of diploid cells. The observed differential staining is discussed in the context of chromatin alterations related to the unique events of meiosis and protein displacement and replacement during sperm differentiation.
Interactive multi‐window integration of two‐parameter flow cytometric data fields
Authors: Kachel Volker 
Published: August 2005

AbstractIntegration is necessary to determine the particle content of regions of interest of flow cytometric two‐parameter fields. The improved program of the Cytomic 12 analyzer (1) offers: window trace integration for relatively simple window structures. The field of interest is surrounded by an integration trace (window). Eight independent windows can be stored and successively evaluated. It also offers painted field integration for complicated window structures. The pointer or a small window is interactively moved over the structures to be integrated like the brush of a painter. The “painted field” defines the window to be integrated. Window sets and painted fields can be stored on a floppy disk. Painted fields can be added and may also serve as look up tables for sorting.
Programming for the DNA analysis of FCM data on an IBM microcomputer
Authors: Pavlik Edward J. , Flanigan Robert C. , Bartmas Jonathan , Van Nagell John R. , Donaldson Elvis S. , Hanson Michael B. , Gallion Holly , Kenady Daniel E. 
Published: August 2005

AbstractThe analysis of data generated on a flow cytometer (FCM) is often performed on a computer obtained especially for dedicated use with the flow cytometer. This computer component can be expensive and also presents the FCM user with the added burden of mastering specialized programming language or of accepting the secret analytical processes of protected proprietary program routines. We believe that the evolution of more accurate and efficient FCM analyses that have the power to consider complex signal distributions can be assisted by the availability of analysis programs written in languages common to many users. DNA analysis routines written for a relatively inexpensive micro‐computer (IBM PC/XT) in Basic and Pascal are described here. The routines can automatically process multiple FCM data files and can provide high‐resolution graphic hardcopy. A foreground/background utilization is also described that allows the computer to be available for other uses in the laboratory.
Flow cytometry of DNA content using oxazine 750 or related laser dyes with 633 nm excitation
Authors: Shapiro Howard M. , Stephens Sandra 
Published: August 2005

AbstractThe laser dyes oxazine 750 (OX750), LD700, and rhodamine 800 (R800) can be used in an instrument employing a low‐power heliumneon laser source for flow cytometry of DNA content in ethanol‐fixed or detergent‐permeabilized cells. Cells in near‐isotonic medium are stained with 10–30 μM dye, and fluorescence excited at 633 nm. The dyes do not appear to stain RNA, and the intensity of DNA staining is not changed when 2 μMHoechst 33342 is added to cells simultaneously with redexcited dye. The effects on fluorescence of addition of DNA to LD700 or R800 in aqueous solution are strongly influenced by the base composition of the DNA; binding mechanisms remain to be determined.
Analysis of protein incorporation of radioactive isotopes in the chinese hamster ovary cell cycle by electronic sorting and gel microelectrophoresis
Authors: Pipkin James L. , Anson Jeanne F. , Hinson William G. , Schol Henry , Burns E. Robert , Casciano Daniel A. 
Published: August 2005

AbstractThe patterns of [3H]‐leucine and [32P]‐phosphate incorporation of proteins extracted with varying molarities of sodium chloride were analyzed from nuclei physically sorted from six fluorescence windows after propidium iodine staining of the G0 + G1 and G2 + M phases of the Chinese hamster ovary (CHO) cell cycle. Eight hundred nanograms of protein were used in each electrophoretic analysis obtained from 200,000 nuclei, a portion of the sample, from each window. Autoradiography was performed in a two‐dimensional polyacrylamide gel ultra‐microelectrophoresis apparatus (UMEA) designed and fabricated in this laboratory. There was a net reduction and/or loss of [3H]‐leucine‐ and [32P]‐phosphate‐labeled protein regions from the autoradiographs occurring primarily in the G2 + M phase. Two phosphorylated proteins that were stage specific were observed in partitions of the G2 + M phase. The use of isolated proteins and the coelectrophoresis of these markers demonstrated the similarity in mobility of a number of proteins seen in the autoradiographs of proteins extracted with high and low salt molarities and implied they are synonymous. Coelectrophoresis indicated that a substantial number of high molecular weight proteins that decreased or disappeared at late stages of G2 + M and early mitosis were composed, in part, of nucleolar proteins.
Quantitation and sorting of vitally stained natural killer cell‐target cell conjugates by dual beam flow cytometry
Authors: Storkus Walter J. , Balber Andrew E. , Dawson Jeffrey R. 
Published: August 2005

AbstractWe have detected formation of stable associations, or conjugates, between fluorescein diacetate‐ (FDA) stained human natural killer (NK) cells and Hoechst 33342‐ (HO342) stained tumor cells by dual laser flow cytometry. Conjugates in mixtures of effectors and targets emitted both green (FDA) and blue (HO342) fluorescence. This was confirmed by cell sorting. More than 90% of the conjugates included one target and one effector cell. Conjugate formation frequency was temperature independent between 4 and 37°C, optimized by 10 min, and stable for 1 hr. Enrichment of effector populations for cells mediating lysis of standard NK targets and for cells reacting with OKM1, Leu‐7, and Leu‐11b monoclonal antibodies also enriched conjugate‐forming cells. Lysis of either OKM1+, Leu‐11b+ effector subpopulations with antibody and complement eliminated, but treatment with these antibodies alone had no effect on, conjugate formation. Effector pretreatment with Leu‐4 or 3A1 and complement increased the frequency of conjugation slightly. Flow‐determined frequencies of NK‐conjugate formation with 14 target cell lines correlated well with data derived from standard microscopic assays. However, the flow method was more rapid, could be used when target and effector were of comparable size, and permitted isolation of conjugates by sorting.