bluuebunny/crossref_metadata_2025_split huggingface dataset:
Preparation and bivariate analysis of suspensions of human chromosomes
Authors: van den Engh G. J. , Trask B. J. , Gray J. W. , Langlois R. G. , Yu L.‐C. 
Published: August 2005

AbstractChromosomes were isolated from a variety of human cell types using a HEPES‐buffered hypotonic solution (pH 8.0) containing KCl, MgSO4, dithioerythritol, and RNase. The chromosomes isolated by this procedure could be stained with a variety of fluorescent stains including propidium iodide, chromomycin A3, and Hoechst 33258. Addition of sodium citrate to the stained chromosomes was found to improve the total fluorescence resolution. High‐quality bivariate Hoechst vs. chromomycin fluorescence distributions were obtained for chromosomes isolated from a human fibroblast cell strain, a human colon carcinoma cell line, and human peripheral blood lymphocyte cultures. Good flow karyotypes were also obtained from primary amniotic cell cultures. The Hoechst vs. chromomycin flow karyotypes of a given cell line, made at different times and at dye concentrations varying over fourfold ranges, show little variation in the relative peak positions of the chromosomes. The size of the DNA in chromosomes isolated using this procedure ranges from 20 to over 50 kilobases. The described isolation procedure is simple, it yields high‐quality flow karyotypes, and it can be used to prepare chromosomes from clinical samples.
Flow cytometric immunofluorescence of rat anterior pituitary cells
Authors: Michael Hatfield J. , Hymer W. C. 
Published: August 2005

AbstractWe have developed a flow cytometric immunofluorescence technique for the quantification of growth hormone (GH), prolactin (PRL), and luteinizing hormone (LH) producing cells. The procedure requires about 24 hours and can objectively count 50,000 cells in about 3 minutes. It is based on indirect‐immunofluorescence (fluorescein) of intracellular hormone using an EPICS V cell sorter. The fluorescein distributions are gated on DNA content (propidium iodide) to eliminate counting cell clumps. Cells from the same suspensions were stained immunocytochemically and counted microscopically (1,000–2,000 cells/sample). Immunofluorescence and immunocytochemistry correlated to within a few percent for GH and PRL cells. Cell suspensions from adult males and females with or without castration and a diethylstilbestrol (DES)‐induced primary pituitary tumor were used to test the method. A major finding of this study was the objective identification of two populations of PRL producing cells, i.e., lightly and intensely stained cells. On the other hand, the fluorescence distribution of PRL cells from DES‐induced pituitary tumors did not fall into two distinct populations but, rather, represented a broad continuum. This method should prove useful in studying the dynamics of pituitary cell populations under various physiological and pharmacological conditions.
Gene mapping by chromosome spot hybridization
Authors: Collard J. G. , de Boer P. A. J. , Janssen J. W. G. , Schijven J. F. , de Jong B. 
Published: August 2005

AbstractA method is described for the localization of cloned single‐copy genes to flow‐sorted chromosomes. Chromosomes were sorted directly onto nitrocellulose filters and the chromosomal DNA was subsequently hybridized with gene‐specific radioactively labeled DNA probes. Mild aspiration of the filters during sorting was applied to collect the deflected chromosomes in a small spot. Sorting of 10,000–30,000 chromosomes was sufficient to detect gene‐specific hybridization with single‐copy DNA probes. Using this technique, we have sublocalized the human c‐myb oncogene to 6q21–q23 by sorting translocated chromosomes with breakpoints in the q21 and q23 region of chromosome 6. Chromosome spot hybridization appears to be a rapid and simple method to assign cloned genes to chromosomes. Hybridization of an unlocalized gene probe to spots of chromosomes pre‐enriched by velocity sedimentation can quickly narrow the choice of chromosomes which need to be sorted. Conversely, individual chromosomes in a flow karyotype can be identified by hybridizing sorted chromosomal DNA with chromosome‐specific DNA probes.
Detection of Escherichia coli in blood using flow cytometry
Authors: Mansour James D. , Robson Jillian A. , Arndt Carolyn W. , Schulte Thomas H. 
Published: August 2005

AbstractA rapid method for the detection of Escherichia coli in blood has been developed. The method employs blood cell lysis, staining of bacteria with ethidium bromide, and detection of stained bacteria using flow cytometry. The detection protocol requires less than 2 h sample handling time and is not dependent on bacterial growth. This method has been applied to human donor blood specimens seeded with various E. coli concentrations and to two rabbit model systems. Bacterial detection is evident from the in vitro human blood studies at levels of 10 E. coli/ml and from in vivo rabbit model studies at < 100 E. coli/ml.
The G0 ⇆ G1 transitions of human lymphocytes as monitored by quantitative 14C‐uridine autoradiography and high‐resolution image analysis
Authors: Giaretti Walter , Abmayr Wolfgang , Dörmer Peter , Santi Leonardo 
Published: August 2005

AbstractHuman lymphocytes, transferred in culture into medium conditioned with phytohemaglutinin, were studied with the combined use of quantitative 14C‐uridine autoradiography and high‐resolution image analysis. During the first 24 h before the onset of DNA synthesis it was possible to study pure G0 and G1 populations. The return of the proliferating cells into the G0 state was clearly monitored in an RNA synthesis rate/DNA content plot during 1‐week growth. On days 3–6, the proliferation decreased progressively, and more cells accumulated in the quiescent G0 state. Geometric, densitometric, and textural parameters evaluated from the Feulgenstained nuclei of the same cells indicated that the decondensation/condensation processes of the nuclear chromatin during the G0 °CG1 transitions paralleled the RNA synthesis activity. The results help to associate descriptors of nuclear morphology, as derived from image analysis, with functional parameters during activation and return to quiescence of human lymphocytes.
A method to estimate the DNA content of whole nuclei from measurements made on thin tissue sections
Authors: Bins M. , Takens F. 
Published: August 2005

AbstractA microdensitometry method that allows estimation of the distribution of the DNA content of nuclei in thin tissue sections is described. The method is based on a theoretical model of Feulgen‐stained spherical nuclei, of different sizes, in each of which the DNA is present as a homogeneous solution. In thin sections of nuclei of different sizes, the fraction of DNA per section is inversely proportional to the radius of the nucleus. Histograms of the product of DNA content and radius per nuclear section are independent of nuclear size but depend on total DNA content. The distribution of the total DNA content of nuclei in a section can be estimated from such a histogram. The results of the measurements of a Feulgen‐stained rat liver section are described.
Flow cytometric analysis of mouse spermatogenic function following exposure to ethylnitrosourea
Authors: Evenson Donald P. , Higgins Paul J. , Grueneberg Dorre , Ballachey Brenda E. 
Published: August 2005

AbstractThe effects of the mutagenic agent ethylnitrosourea (ENU) on spermatogenic function and sperm chromatin structure were studied by flow cytometry and the results compared with sperm head morphology measurements. Groups of mice received daily exposures ranging from 0 to 75 mg/kg body weight °C5 days and were sacrificed 28 days later. Fresh testicular cell suspensions and epididymal sperm were stained with acridine orange (AO) and measured by flow cytometry. Sperm nuclei were isolated, fixed, rehydrated, and then either subjected to thermal stress or not prior to staining with AO. Body weights were unaffected by the chemical exposure while the testicular weights were reduced by about 50%. Two‐parameter (DNA, RNA) flow cytometry measurements showed a dose‐response relationship in the loss of certain cell types, particularly the elongated spermatids, from the testes of treated animals. Flow cytometric analysis of both heat‐stressed and non‐heat‐stressed nuclei showed a relationship between dosage and the coefficient of variation of α t {red/(red + green fluorescence)} measurements of AO stained nuclei, thereby demonstrating that alterations of chromatin structure occurred in response to ENU. Enzymatic digestions with RNAse, DNAse, and nuclease S1 suggest that the increase in red fluorescence is due to an increase of single‐stranded DNA induced by heat or acid treatment of chemically altered chromatin structure. The lowest daily dosage used (5 mg/kg) caused no significant changes in ratios of testicular cell types, a questionable increase in abnormal sperm head morphology and a detectable change in chromatin structure expressed as α t. This report shows that our technique for assaying sperm nuclear chromatin structure appears to have the same level of sensitivity to ENU induced nuclear alterations as the sperm head morphology test.
The use of flow cytometry and cell sorting in a human colon carcinoma model
Authors: Noguchi Philip D. , Cunningham Robert E. , Ridge Jeanette , Muller Jacqueline 
Published: August 2005

AbstractWe investigated the growth characteristics of a human colon carcinoma cell line, WiDr, grown in culture flasks and on chick embryonic skin (CES). WiDr cells labeled in vitro with bromodeoxyuridine (BrdU) and analyzed by combined propidium iodide/Hoechst 33258 fluorescence showed evidence of more BrdU incorporation in early S phase as compared to late S phase. When inoculated on the CES, WiDr cells multiplied and invaded the underlying skin. Morphologic examination showed that with extended culture WiDr cells on the CES undergo progressive structural organization with the development of acini and basal lamina, structures similar to those in in vivo tumors. WiDr cells were labeled with monoclonal antibody to carcinoembryonic antigen (CEA) and the brightest 2% of the population was sorted. When subsequently grown on the CES, the sorted cells formed significantly more acinar structures at 3 and 6 days of culture than an unsorted population grown for a comparable time.
Flow cytometric analysis of fluorescein‐conjugated estradiol (E‐BSA‐FITC) binding in breast cancer suspensions
Authors: Benz Chris , Wiznitzer Israel , Lee Sin Hang 
Published: August 2005

AbstractWith greater utilization of histochemical methods for detecting estrogen binding (ER) in tumor cells, there is an increasing need to quantitate objectively these fluorescently stained cells. This study utilizes flow cytometry (FCM) to examine the binding specificity and kinetics of 17β‐estradiol‐6‐CMO‐BSA‐FITC (E‐BSA‐FITC) in two human mammary carcinoma cell lines, MCF‐7 and 47‐DN. Cells are rendered permeable to this ligand by freeze‐thawing, a process analogous to the routine staining of frozen tumor sections with E‐BSA‐FITC for the clinical detection of ER. FCM quantification of E‐BSA‐FITC binding intensity demonstrates a saturable dose‐reponse that is specifically reduced in the presence of diethylstilbestrol (DES) in doses known to saturate Type I ER. Scatchard analysis suggests that E‐BSA‐FITC binding occurs with receptors of varying affinities (Kd). Lineweaver‐Burk plots show that the DES inhibition is competitive for a high‐affinity receptor binding to E‐BSA‐FITC with a Kd of approximately 50 nM.This report also compares both FCM and biochemical methods of quantitating ER under two conditions of tumor cell growth potentially encountered in clinical specimens: quiescent versus actively proliferating cells, and cells pretreated with the antiestrogen tamoxifen. By FCM analysis, the cells with greater proliferating activity contain tenfold more specifically bound E‐BSA‐FITC, and tamoxifen pretreatment reduces this specific binding by 50%. These FCM measurements correlate well with biochemical results and suggest that this new methodology may supplement the detection of E‐BSA‐FITC binding by fluorescence microscopy.
Dual laser flow cytometry: Focal length compensation when focussing through a single lens
Authors: Fellner‐Feldegg Hugo 
Published: August 2005

AbstractDual laser operation of flow cytometers, using a single focussing lens for both beams, requires compensating for chromatic aberration of the lens. By using a prefocussing lens at a fixed position in one of the laser beams, complete focal length compensation is obtained without any loss in system performance.
The LLNL high‐speed sorter: Design features, operational characteristics, and biological utility
Authors: Peters D. , Branscomb E. , Dean P. , Merrill T. , Pinkel D. , Van Dilla M. , Gray J. W. 
Published: August 2005

AbstractThis report describes a dual‐beam, high‐speed sorter (HiSS) with a droplet production rate of 220,000 s−1 now in routine use at the Lawrence Livermore National Laboratory. The system can process and sort objects at rates in excess of 20,000 s−1. We report here on the development of HiSS, describe its operational characteristics, and evaluate its utility for analysis and purification of human chromosomes, human erythrocytes, and living Chinese hamster ovary cells.
Flow cytometric fluorescence emission spectrum analysis of hoechst‐33342‐stained DNA in chicken thymocytes
Authors: Watson James V. , Nakeff Alexander , Chambers Stephen H. , Smith Paul J. 
Published: August 2005

AbstractHoechst‐33342‐stained chicken thymocytes were analysed simultaneously on two fluorescence wavelength bands (green and violet) in our custom‐built flow cytometer, and two major subsets were identified. In one subset (33% of the total) the emission spectrum remained constant with time, with little change in the respective green and violet fluorescence intensities. In the other subset (42% of the total) the green fluorescence increased during staining, resulting in a considerable change in the green‐to‐violet ratio, due to a change in the “shape” of the fluorescence emission with time. The data indicate that two binding sites, or two types of binding at the same site, exist in DNA for this dye and that these have different binding energies and, consequently, different fluorescence emission properties.
DNA analysis and sorting of rat testis cells using two‐parameter flow cytometry
Authors: van Kroonenburgh Marinus J. , Beck Johannes L. , Scholtz Johannes W. , Hacker‐Klom Ursula , Herman Chester J. 
Published: August 2005

AbstractBy use of two‐parameter flow cytometry of rat testis cell suspensions stained with mithramycin for DNA (the peak amplitude of the fluorescence signal versus total fluorescence intensity integrated over time), eight cell compartments could be distinguished without pre‐enrichment of the samples. Cells in these compartments were identified by sorting and subsequent microscopic examination.
DNA distribution in non‐small‐cell lung carcinomas and its relationship to clinical behavior
Authors: Volm Manfred , Mattern Jürgen , Sonka Jaroslav , Vogt‐Schaden Marlies , Wayss Klaus 
Published: August 2005

AbstractA study of 187 surgical specimens of tumors of patients with non‐small‐cell lung carcinomas was carried out by means of flow cytometry. Eighty‐four percent of the tumors were classified as tumors with abnormal DNA stemlines (DNA aneuploidy). Patients with tumors demonstrating DNA aneuploidy had significantly shorter survival times than those with tumors demonstrating DNA diploidy (p = .009).Cell cycle analysis was possible in 122 tumors. Patients whose tumors had 0–8% S‐phase cells died later than patients whose tumors had 9–16% S‐phase cells (p = .018). In addition, patients with tumors with a low fraction of labeled S‐phase cells (autoradiography) had a better prognosis than patients with tumors with a high proportion of labeled S‐phase cells (p = .041).
Short‐term cryopreservtion of human breast carcinoma cells for flow cytometry
Authors: Stone Kenneth R. , Bruce Craig R. , Palmer James O. , Rivkin Saul E. , McDivitt Robert W. 
Published: August 2005

AbstractA procedure is described for short‐term cryopreservation of primary human tumor cells and tissue slices for later analysis by flow cytometry. Cells were mechanically dispersed into a freezing medium, which was then frozen at either –20°C or –70°C for delayed cell cycle analysis. The results show that a correlation coefficient of greater than 0.95 exists between cell cycle kinetic analyses performed immediately after surgical excision of the tumor and on cells frozen from 1 to 30 days at –70°C in this freezing solution. Somewhat lower levels of correlation exist for cells frozen at –20°C in this freezing medium. This procedure has also been successfully used to preserve freshly isolated breast carcinoma cells shipped from distant laboratories for analysis in the flow cytometer, thus expanding the data base on certain types of breast carcinoma.
Cell kinetics in normal and neoplastic mammary tissues by flow cytometric analyses
Authors: Kute Timothy E. , Linville Constance , Mehta Rajendra G. , Moon Richard C. 
Published: August 2005

AbstractThe cell cycle kinetics of normal mammary tissue and cancer mammary tissues were studied by flow cytometry to determine the effect of estrogen and progesterone and antiestrogens on proliferative activity. The proliferative activity was higher in rat mammary tissue during the estrus and pregnant stages compared to the nonestrus or lactational stages. Upon 5 days administration of estrogen and progesterone to the lactating animal, the low proliferative activity was increased to the values found in the pregnant stage. Steroidal effect on proliferative activity was also shown using organ cultures of normal rat tissues and the increase in proliferative activity can be reversed by the addition of 10−6 M retinoic acid but not by retinyl acetate. In a human mammary tumor cell line (MCF‐7), the cell kinetic activity was significantly depressed by antiestrogens (i.e., tamoxifen) as determined by both flow cytometry and tritiated thymidine uptake studies. Finally, a human tumor model system was developed with the MCF‐7 cell line in estrogenized nude mice. Flow cytometry of the resulting tumor indicates a tetraploid tumor similar to the cell culture. Cell kinetic analysis of these nude mice tumor cells demonstrates the higher proliferative activity of the estrogenized animals compared to the tamoxifen‐treated animals.In conclusion, the use of flow cytometry to rapidly measure cell kinetic activity of mammary tissue has been demonstrated. The effects of estrogen or estrogen and progesterone hormones seem to increase the proliferative activity, while antiestrogens decrease this activity. Findings are observed in both in vivo and in vitro conditions or normal tissues, mammary cancer tissue, and cultured cells.
Automated analysis and survival selection of anchorage‐dependent cells under normal growth conditions
Authors: Schindler Melvin , Allen Margaret L. , Olinger Max R. , Holland John F. 
Published: August 2005

AbstractAn instrument is described that can automatically analyze and select for a subpopulation of anchorage‐dependent cells in tissue culture. Cells that label with fluorescently tagged antibodies or demonstrate structural variations are saved from exposure to a destructive high‐intensity argon laser beam. The surviving population may then be cloned. The cell selection may occur in a tissue culture plate or in a microflow incubator which is designed to maintain a constant flow of media at 37°C across cells growing on a glass coverslip. This incubator sits on an inverted microscope which focuses the laser beam to a diameter as small as 1 μm. A high‐speed computer‐controlled two‐dimensional stage moves the cells past the beam for analysis, the results of which determine the fate of each cell: whether it is to be destroyed by radiant energy or selected for survival and subsequent proliferation. Another selection strategy performed by the instrument involves growing the cells on a thin, blackened polyester film which can be cut by the argon laser beam. Cells selected for cloning are then circumscribed. The heat of cutting welds the circumscribed film to a plastic coverslip surface or tissue culture chamber bottom. Nonselected cells may be removed by pulling the unattached polyester sheet from the attachment surface. The selected cells remain on polyester film disks welded to the plastic. Selections may be done automatically under computer control or manually by operator direction of stage movements.This instrument extends the art of automated cell selection and analysis to normal cell lines that must maintain cell‐substratum contact (anchorage dependence) for differentiated cell function, e.g., neurons, fibroblasts, or kidney cells.
RNA content and chromatin structure of CHO cells arrested in metaphase by colcemid
Authors: Melamed Joseph , Darzynkiewicz Zbigniew 
Published: August 2005

AbstractTo evaluate the stability of cells arrested in metaphase, cell viability, RNA content, and chromatin structure (the latter probed by the DNA in situ sensitivity to acid‐induced denaturation) were studied in uniform‐age mitotic CHO cell populations maintained either at 37°C (in the presence of Colcemid) or at 0–4°C for up to 6 h. No significant changes in cell viability and RNA content were seen throughout the experiment for both groups of cells. The sensitivity of DNA in situ to denaturation was significantly increased during the initial 40 min of cell arrest in mitosis. However, no further chromatin changes for up to 6 h were evident regardless of whether cells were kept at 37°C with Colcemid or at 0–4°C in its absence. The data indicate that neither significant deterioration of metaphase cells nor progressive chromatin changes are expected during stathmokinesis experiments in vitro or during the metaphase cell arrest in cytogenetic studies lasting up to 6 h. Also, no RNA turnover can be detected in mitotic cells during this time interval.
Evaluation of a new blood cell counter with sheath flow system
Authors: Tatsumi Noriyuki , Tsuda Izumi , Fujimoto Keiji , Matsumoto Hideaki 
Published: August 2005

AbstractA blood cell counter with a sheath flow system has been developed to eliminate the drawback of the Coulter type blood cell counter, namely, signal distortion caused by the cells passing through the electric aperture gate. With the new system, signal distortion was low and a near‐normal distribution curve for erythrocytes and resin particles was obtained. The counter has a computer program for determining the red cell distribution width, which represents an actual size difference at 20% of the relative frequency of the distribution curve. This is independent of mean cell volume, and is considered to be of clinical importance. We examined the values in 2,300 healthy subjects and patients with various hematological disorders.
A dual laser analysis of the migration of XRITC‐labeled, FITC‐labeled, and double‐labeled lymphocytes in sheep
Authors: Abernethy Nevin J. , Chin Warren , Lyons Helen , Hay John B. 
Published: August 2005

AbstractSubstituted rhodamine isothiocyanate (XRITC) has been used to study lymphocyte migration in sheep. After being labeled in vitro with XRITC, lymphocytes appeared in the efferent lymph of single lymph nodes with the same kinetics as cells labeled with fluorescein isothiocyanate (FITC). The recovery of intravenously injected XRITC‐labeled cells was followed in lymph for several days. The kinetics and recoveries were compared with data obtained using FITC, chromium‐51, and indium‐111. XRITC was found to be a suitable label and, using dual laser (argon and krypton) flow cytometry, it could be analyzed simultaneously with FITC. In addition, it was possible to relabel FITC‐stained cells with XRITC after they were recovered in lymph. The migratory characteristics of such doublelabeled cells were not different from singlelabeled cells.