bluuebunny/crossref_metadata_2025_split huggingface dataset:
Image analysis in comparative genomic hybridization
Authors: Lundsteen Claes , Maahr Jan , Christensen Britta , Bryndorf Thue , Bentz Martin , Lichter Peter , Gerdes Tommy 
Published: June 2005

AbstractComparative genomic hybridization (CGH) is a new technique by which genomic imbalances can be detected by combining in situ suppression hybridization of whole genomic DNA and image analysis. We have developed software for rapid, quantitative CGH image analysis by a modification and extension of the standard software used for routine karyotyping of G‐banded metaphase spreads in the Magiscan chromosome analysis system. The DAPI‐counterstained metaphase spread is karyotyped interactively. Corrections for image shifts between the DAPI, FITC, and TRITC images are done manually by moving the three images relative to each other. The fluorescence background is subtracted. A mean filter is applied to smooth the FITC and TRITC images before the fluorescence ratio between the individual FITC‐ and TRITC‐stained chromosomes is computed pixel by pixel inside the area of the chromosomes determined by the DAPI boundaries. Fluorescence intensity ratio profiles are generated, and peaks and valleys indicating possible gains and losses of test DNA are marked if they exceed ratios below 0.75 and above 1.25. By combining the analysis of several metaphase spreads, consistent findings of gains and losses in all or almost all spreads indicate chromosomal imbalance. Chromosomal imbalances are detected either by visual inspection of fluorescence ratio (FR) profiles or by a statistical approach that compares FR measurements of the individual case with measurements of normal chromosomes. The complete analysis of one metaphase can be carried out in approximately 10 minutes. © 1995 Wiley‐Liss, Inc.
Semiautomated DNA probe mapping using digital imaging microscopy: II. System performance
Authors: Sakamoto Masaru , Pinkel Daniel , Mascio Laura , Sudar Damir , Peters Donald , Kuo Wen‐Lin , Yamakawa Kazuhiro , Nakamura Yusuke , Drabkin Harry , Jericevic Zeljiko , Smith Louis , Gray Joe W. 
Published: June 2005

AbstractThis paper describes an evaluation of a semiautomated, multicolor image‐analysis system to map cloned probes along metaphase chromosomes. Mapping with this system consists of fluorescence in situ hybridization (FISH) for probe localization, automatic acquisition of multicolor images showing total chromosomal DNA and probe location(s), and automatic determination of the fractional locations of the probes along the chromosomes relative to the short arm telomere (FLpter). The system was evaluated by mapping ten phage and ten cosmid probes previously mapped to chromosome 3 with other procedures. The standard deviations of FLpter measurements averaged 3.4 Mb and 2.6 Mb for phage and cosmid probes, respectively. With this variation, the order of two probes mapped in separate hybridizations could be determined with 95% confidence when their separation was greater than 2.5 Mb. In all cases, the probe locations and order were consistent with previous mapping data. FLpter values were converted to band locations using measurements of the band locations made using digital imaging microscopy. This proved superior to conversions made using ISCN ideograms. © 1995 Wiley‐Liss, Inc.
Flow injection fluorescence microscopy applied to a rapid cell surface immunoassay
Authors: Pollema Cy H. , Lernmark Åke , Ruzicka Jaromir 
Published: June 2005

AbstractA perfusion system for fluorescence microscopy that utilized a flow injection system was developed and used to study cell surface antibody binding on viable cells grown in monolayer cultures on coverslips. A polyclonal cell‐specific antiserum used to probe the cell surface was monitored by indirect immunofluorescence. The flow injection system was completely automatic and allowed controlled perfusion of cell surfaces with the desired sequence of antibodies. The individual steps of the indirect assay were studied to determine the binding behavior of the primary cell surface antibody, the labeled second antibody, and control of nonspecific binding. Under stopped flow conditions, the second antibody was maximally bound within 10 min, while constant mixing of the second antibody solution over the cells resulted in maximal binding within as little as 6 min. A primary antibody contact time of 2 min followed by a wash and then exposure to the second antibody for 2 min showed that this fast automated procedure could distinguish specific from nonspecific binding of cell surface antibodies to the same set of cells for several repeated exposures. The flow injection fluorescence microscopy system can be automated to allow screening for cell surface antibodies and to study their interaction with specific cell surface antigens. © 1995 Wiley‐Liss, Inc.
Flow cytometric assessment of peripheral blood contamination and proliferative activity of human bone marrow cell populations
Authors: Abrahamsen Jenny Foss , Lund‐Johansen Fridtjof , Laerum Ole Didrik , Schem Baard Christian , Sletvold Olav , Smaaland Rune 
Published: June 2005

AbstractBone marrow aspiration is superior to bone marrow biopsies due to less discomfort to the volunteer or patient, but it is inferior concerning the reproducibility of cytokinetic information. Therefore, a method that could select aspirates of quality and reproducibility equal to those of biopsies was sought. Low‐density (mononucleated) bone marrow cells were labelled with T200 common leukocyte antigen, CD45, which differentiate cells into erythroid, myeloid, and lymphocyte + monocyte subpopulations based on their immunofluorescence intensity. A hypotonic propidium iodide solution was added, and DNA cell cycle characteristics of the total cells and the subpopulations were obtained. Twenty‐two aspirations were performed on three healthy men. There was a strong negative correlation between the amount of CD45‐gated lymphocytes + monocytes, indicative of peripheral blood cell contamination in the aspirate, and the percentage of total cells and subpopulations in DNA S phase. A marked reduction in the percentage of cells in S phase was observed when the lymphocyte + monocyte counts were higher than 30%; this level was used to exclude aspirates with an unacceptable degree of peripheral blood cell admixture. Twelve of the aspirates were found to be of acceptable quality due to their low lymphocyte + monocyte count. These aspirates were compared with 11 bone marrow biopsy expellates from hematologically normal patients undergoing open cardiac surgery. The 12 aspirates were found to have almost identical mean percent S‐phase cells as the biopsy expellates, both for the total cell population (14% ± 3.45% vs. 15% ± 1.5%) and for the erythroid (24% ± 6% vs. 24.4% ± 3.3%) and myeloid (10% ± 2.4% vs. 10.7% ± 2.5%) subpopulations. The aspirates with an unacceptable degree of peripheral blood cell admixture had a significantly lower mean percent cells in S phase for both the total cells and the subpopulations. It is concluded that bone marrow aspirates of small volumes may give cytokinetic information as reproducible as bone marrow biopsies if the above‐described method is applied. © 1995 Wiley‐Liss, Inc.
Classmode: A new data format for real‐time multiparameter data analysis and data compression
Authors: Keij Jan F. , Groenewegen Ad C. , Jonker Richard R. , Smith Chris R. , Visser Jan W. M. 
Published: June 2005

AbstractA new data acquisition and analysis format (class‐mode) was developed that allows real‐time data classification in a flow cytometer. In our cytometer, detected events were classified in real time by their presence or absence in a set of look‐up tables (LUT). A modification of the cytometer hardware allows the exclusive transfer of the LUT data to the acquisition/storage computer. Using a combination of 8 LUTs, the analyzed events can be classified into 256 subpopulations. Real‐time data classification results in an increased data transfer rate and a significant compression of the data. © 1995 Wiley‐Liss, Inc.
Sex determination in dioecious plants Melandrium album and M. rubrum using high‐resolution flow cytometry
Authors: Doležel Jaroslav , Göhde Wolfgang 
Published: June 2005

AbstractHigh‐resolution flow cytometry was used to analyze nuclear DNA content in dioecious plants Melandrium album and M. rubrum. Very low coefficients of variation (0.53–0.70%) of G0/1 peaks were obtained after the analysis of nuclei isolated by chopping leaf tissues. In both species, females have lower DNA content than males due to sex chromosome heteromorphism. This made it possible to discriminate peaks belonging to female and male nuclei on the same histogram and permitted unambiguous sex identification in both species. The results indicated the possibility of diagnosing sex also in other heterogametic dioecious plants. The assay described here might be interesting in any area where rapid sex identification is required at an early stage of plant development. © 1995 Wiley‐Liss, Inc.
Expression of HSP70 induced in CHO cells by 45.0°C hyperthermia is cell cycle associated and DNA synthesis dependent
Authors: Hang Haiying , Fox Michael H. 
Published: June 2005

AbstractIn response to a heat shock, the synthesis of heat shock proteins with a molecular weight of 72 kD (hsp72) is induced. To determine whether hsp72 expression in Chinese hamster ovary (CHO) cells is cell cycle associated, flow cytometry was used to measure DNA content and heat shock protein levels simultaneously. After 10 min at 45.0°C and incubation at 37°C for several hours, hsp72 was induced to a high level in early S phase. Inhibitors of DNA synthesis, aphidicolin, and 1‐β‐D‐arabinofuranosylcytosine (araC), suppressed the induction of hsp72 but did not inhibit it completely. Hsp73 (constitutive hsp70) was also induced by heat shock, but the induction was uniform through the cell cycle and it was not suppressed by araC. © 1995 Wiley‐Liss, Inc.
Cell cycle analysis of amount and distribution of nuclear DNA topoisomerase I as determined by fluorescence digital imaging microscopy
Authors: Baker Sharyn D. , Wadkins Randy M. , Stewart Clinton F. , Beck William T. , Danks Mary K. 
Published: June 2005

AbstractFluorescence digital imaging microscopy (FDIM) has been used to perform a cell cycle analysis of both the amount and the distribution of nuclear DNA topoisomerase I in individual CEM human leukemia cells. Cells were stained by indirect immunofluorescence methods using a polyclonal antiserum generated with a 21‐amino‐acid peptide representing amino acids 219–239 of human topoisomerase I. Immunohistochemical staining was followed by staining with Hoechst dye 33342, allowing DNA content to be determined in each cell. Cell cycle analysis showed that nuclear topoisomerase I content doubled (2.2‐fold increase) as the cells progressed from G1 to G2/M phases of the cell cycle. However, when normalized for nuclear size, topoisomerase I content per nuclear area remained almost constant (1.3‐fold increase). For comparison, we measured the amount of proliferating cell nuclear antigen (PCNA), a protein whose expression fluctuates during the cell cycle. Nuclear PCNA content increased 2.7‐fold from G1 to S phase, then declined in G2/M‐ phases, whereas PCNA content per nuclear area increased 1.7‐fold from G1 to S phase. We also measured topoisomerase I content in leucine‐deprived cells to determine if altered growth conditions affect topoisomerase I protein expression. Compared to CEM cells in logarithmic growth, leucine‐deprived CEM cells had 1.8‐fold less topoisomerase I content per nuclear area. Subnuclear distribution studies of proliferating CEM cells showed topoisomerase I to be localized predominantly in the nucleoli throughout the cell cycle. In contrast, leucine‐deprived cells exhibited a perinuclear distribution of topoisomerase I. Our results show that FDIM is a useful technique in determining the cell cycle position and both the content and the distribution of topoisomerase I as well as other nuclear proteins in individual cells. © 1995 Wiley‐Liss, Inc.
Analysis of antifading reagents for fluorescence microscopy
Authors: Florijn R. J. , Slats J. , Tanke H. J. , Raap A. K. 
Published: June 2005

AbstractThe utility of p‐phenylenediamine, 1,4‐di‐azobicyclo‐(2.2.2.)‐octane, and the commercial products Citifluor, Slowfade, and Vectashield, antifading agents frequently used as mounting media for fluorescence in situ hybridization, was investigated. Fading curves for bound fluorochromes were recorded with digital microscopy, and relative fluorescence intensities of fluorochromes in solution were measured with an aperture defined measurement system. The three commonly used fluorochromes, fluorescein, tetramethyl rhodamine, and coumarin, were studied. Vectashield offered the best antifading properties for all three fluorochromes, although their relative fluorescence intensity was slightly less in Vectashield in comparison with other antifading agents. In Vectashield, fluorescein, tetramethyl rhodamine, and coumarin showed half‐life times of 96, 330, and 106 s, respectively, whereas in 90% glycerol in PBS (pH 8.5), these half‐life time values were 9, 7, and 25 s, respectively. Vectashield is particularly recommended as a mounting medium for quantitative digital imaging microscopy and for multicolor applications, where it is easy to have errors due to differences in fading rates of the fluorochromes. © 1995 Wiley‐Liss, Inc.
Usefulness of the scraping method for DNA flow cytometry in breast tumors
Authors: Cornacchiari A. , Grigolato P. G. , Facchetti F. , Morassi M. L. , Cadei M. , Alpi F. , Battocchio S. , Chirico E. 
Published: June 2005

AbstractFlow cytometric DNA analysis is an important prognostic tool in breast cancer. We evaluated the possibility of performing DNA analysis on cell suspensions obtained by scraping the cut surface of breast tumors; 31 breast tumor nodules, including six benign and 25 malignant lesions, were studied. From each case, cell suspensions acquired by mechanical mincing of a fresh frozen tissue fragment and by two different scrapings (central and peripheral) from the cut surface of the tumor were analyzed via flow cytometry. In all cases, comparison of the DNA histograms for three samples showed no significant differences in the appearance of debris or in the value of coefficient of variation of the G0–G1 peak. All benign nodules showed a normal DNA stemline in all specimens. In 23 of 25 cases of breast carcinoma, the ploidy of the three preparations was similar, with a concordance in 12/14 (85, 71%) cases in DNA nondiploid tumors. Linear regression analysis showed a good correlation in DNA index between either scraping sample and the tissue fragment (r = .955 and r = .905). The results indicate that the scraping technique provides excellent cell suspensions and DNA histograms comparable to those obtained from mechanical mincing of tissue fragments. The technique minimizes preparation time and avoids consuming much tissue and, thus, is the method of choice when very small cancers have to be analyzed. © 1995 Wiley‐Liss, Inc.
Comparative sperm chromatin structure assay measurements on epiillumination and orthogonal axes flow cytometers
Authors: Evenson Donald , Jost Lorna , Gandour Donna , Rhodes Lori , Stanton Barbara , Clausen Ole Petter , de Angelis Paula , Coico Richard , Daley Andrew , Becker Kevin , Yopp Tim 
Published: June 2005

AbstractThe sperm chromatin structure assay (SCSA) measures the susceptibility of sperm nuclear DNA to acid‐induced denaturation in situ, and was developed on two Ortho flow cytometers, an FC200 [Becton Dickinson Immunocytometry Systems (BDIS), Westwood, MA] and a Cytofluorograf 30 (BDIS), both having orthogonal axes of fluorochrome excitation, emission, and sample flow. Sperm cells are first treated with a pH 1.4 buffer to denature DNA in situ and then stained with the metachromatic dye acridine orange (AO). The metachromatic fluorescence measured reflects relative amounts of denatured (red fluorescence) and native (green fluorescence) DNA present per cell. The extent of DNA denaturation is quantified by the calculated parameter alpha t [αt = red/(red + green) fluorescence]. Alpha t variables important for correlations with fertility and toxicant‐induced chromatin damage include mean (Xαt), standard deviation (SDαt), and cells outside the main population (COMPαt). Mean green fluorescence intensity is an important measure for DNA content and/or degree of sperm chromatin condensation. This study showed that the SCSA can be successfully run on two epiillumination‐type instruments, an Ortho ICP22A (BDIS, San Jose, CA) and Skatron ArgusTM (Tranby, Norway), and two additional orthogonal axes instruments, a Becton Dickinson FACScanTM (BDIS) and a Coulter EliteTM (Coulter Corporation, Hialeah, FL). Epiillumination instruments produced a different fluorescence distribution than orthogonal instruments, but the resulting αt values showed strong conformity and interpretation of results was the same. SCSA values obtained on the Coulter EliteTM were most similar to the Cytofluorograf 30; the FACScanTM green fluorescence distribution was narrower and allowed resolution of cell doublets. Neither orthogonal instrument has the ability to directly calculate αt values Listmode data from these instruments were transferred to an off‐line personal computer (PC) for calculation of αt values using LIST‐VIEWTM software (Phoenix Flow Systems, Inc., San Diego, CA). © 1995 Wiley‐Liss, Inc.
Induction of cell cycle perturbations by the tear gas 2‐chlorobenzylidene malonitrile in synchronously and asynchronously proliferating mammalian cells
Authors: Weller Eva Maria , Kubbies Manfred , Nüsse Michael 
Published: June 2005

AbstractThe effects of the tear gas 2‐chlorobenzylidene malonitrile (CS) on mammalian cell proliferation were studied in detail using bromodeoxyuridine/ Hoechst flow cytometry. In synchronized (G0/G1‐phase) Chinese hamster embryo (CHE) cells, exposure to CS (60 μM) caused a permanent arrest in the G0/G1 phase in 50% of the cells and a delayed G0/G1 phase exit. In asynchronously growing CHE cells, the CS‐induced cell kinetic perturbations varied with the cell cycle stage during treatment. While G1‐phase cells showed a delayed progression through S and G2/M phases, S‐phase cells were mainly inhibited in the G2/M compartment of the first cell cycle. In contrast, CS‐treated, asynchronous, amniotic fluid‐derived, fibroblast‐like (AFFL) cells exhibited a prolonged transit through the G2/M phase of the first cell cycle regardless of the cell cycle stage during treatment. This indicates that the induced cytotoxicity of CS is a function of both the cell cycle phase and the particular type of cells. © 1995 Wiley‐Liss, Inc.
Phenotype transition of CD4+ T cells from CD45RA to CD45RO is accompanied by cell activation and proliferation
Authors: Johannisson Anders , Festin Roger 
Published: June 2005

AbstractAn investigation of proliferation and activation events in subsets of human CD4+ cells, defined by their expression of CD45RA and CD45R0, is reported. A single‐laser based assay for the study of multiple surface antigens and two‐parameter cell cycle analysis was used for sorting of and subsequent analysis of proliferation in CD4+ CD45RA+ CD45R0−, CD4+CD45RA−CD45R0+ subsets and phenotypically intermediate stages. After labelling with BrdUrd, cells were sorted with flow cytometry on the basis of light‐scattering properties and staining with anti‐CD45RA, anti‐CD45R0, and anti‐CD4 markers. Sorted cells were double stained with anti‐BrdUrd‐antibodies and PI, and the frequencies of proliferating cells were determined. After 48 h, the highest rate of proliferation was found among cells with a phenotype intermediate between CD4+ CD45RA+CD45R0− and CD4+CD45RA−CD45R0+. After 72 h of culture, the situation was changed insofar as the point of highest proliferation had shifted towards the CD4+CD45RA−CD45R0+ population. These findings were further corroborated by four‐colour staining with anti‐CD4, anti‐CD45RA, anti‐CD45R0, and Hoechst 33342. This indicates that the phenotype transition is accompanied by cell proliferation. The correlated temporal expression of antigens related to activation (HLA‐DR, CD25, CD69, CD71) and cell adhesion (CD11a, CD54, L‐selectin) in each of the different subsets was also investigated. All the activation markers CD25, CD69, and CD71 show a more heterogeneous pattern of expression among the CD4+ CD45RA−CD45R0+ cells than the CD4+ CD45RA+CD45R0− cells, indicating a subpopulation of CD4+CD45RA−CD45R0+ cells responding more slowly to the mitogenic stimulation. © 1995 Wiley‐Liss, Inc.
Flow cytometric evaluation of erythrocyte response to oxidant stress
Authors: Hammouda Abd‐El‐Rahman M. A. , Fakeir Aisha 
Published: June 2005

AbstractErythrocyte response to oxidant challenge has been used as an indicator of cell membrane tolerance to oxidant stress. This study describes a simple technique for evaluation of erythrocyte response to hydrogen peroxide oxidant stress using flow cytometry as an assay method. Challenged cells showed detectable increase in fluorescence over the basalevel. This increase in fluorescence was both concentration and time dependent. The test was standadized using 0.1 mM hydrogen peroxide and 2 h incubation. The standardized test showed a higher fluorescence of challenged umbilical cord erythrocytes compared to challenged adult red blood cells. We recommend further use of this test for examining other populations of red blood cells. © 1995 Wiley‐Liss, Inc.
Specific staining of iododeoxyuridine and bromodeoxyuridine in tumors double labelled in vivo: A cell kinetic analysis
Authors: Pollack Alan , Terry Nicholas H. A. , Wu Catherine S. , Wise Birute M. , White R. Allen , Meistrich Marvin L. 
Published: June 2005

AbstractThe simultaneous and specific staining of iododeoxyuridine (IdUrd) and chlorodeoxyuridine (CldUrd) allows for more accurate estimates of potential doubling time (Tpot). Because CldUrd is not approved for human use, the procedure was adapted for the staining of IdUrd and bromodeoxyuridine (BrdUrd). The fluorescein isothiocyanateconjugated B44 antibody (B44‐FITC) stained both IdUrd and BrdUrd in tumor nuclei labelled singly with one or the other pyrimidine analogue. However, when MCaK tumors in exponential growth in vivo were pulse labelled with both IdUrd and BrdUrd, the staining of BrdUrd was not seen, and the labelling pattern reflected specificity to IdUrd. These observations were confirmed using tumors pulse labelled with IdUrd and/or BrdUrd at 6 h and/or 0.3 h prior to tumor removal in all possible combinations. Simultaneous specific staining of BrdUrd by Br3 and of IdUrd by B44‐FITC was documented by quantification of labelling indices (LIs) from double‐labelled tumors. The specificity of B44‐FITC for IdUrd in double‐labelled tumors was due to a greater affinity of this antibody for IdUrd than for BrdUrd. This technique allowed for two independent estimates of LI and Tpot when tumors were double labelled for 3.0 and 5.5 h. Both IdUrd and BrdUrd are approved for clinical use, and this double‐labelling technique should prove to be valuable for measuring the cell kinetics of solid tumors in vivo. © 1995 Wiley‐Liss, Inc.