bluuebunny/crossref_metadata_2025_split huggingface dataset:
Tumor cell kinetics using two labels and flow cytometry
Authors: Ritter Mark A. , Fowler John F. , Kim Youngmi J. , Gilchrist Kennedy W. , Morrissey Lawrence W. , Kinsella Timothy J. 
Published: June 2005

AbstractA flow cytometry based, single sampling method employing sequential bromodeoxyuridine (BrdUrd) and iododeoxyuridine (IdUrd) labeling with an intervening interval is examined. BrdUrd‐specific and nonspecific monoclonal antibodies and flow cytometry are used to estimate the duration of DNA synthesis and the potential doubling time in tumor cell populations using a single sampling or biopsy. A correction for labeled, divided cells allows postlabel incubation intervals to exceed the length of the G2/M phase of the cell cycle. The method yields reliable results when tested in experimental tumor systems in vitro and in situ, as well as in nine human tumors labeled in situ. It uses a somewhat simpler analysis than the alternative relative movement method, requiring only labeling indices, rather than both a labeling index and relative movement, but does require the administration of two, rather than one, label. It provides an independent verification of and a useful single sampling alternative to the relative movement method for the estimation of the length of S phase and, from this, the potential doubling time in experimental or clinical human tumors. © 1994 Wiley‐Liss, Inc.
Slit‐scanning technique using standard cell sorter instruments for analyzing and sorting nonacrocentric human chromosomes, including small ones
Authors: Rens W. , Van Oven C. H. , Stap J. , Jakobs M. E. , Aten J. A. 
Published: June 2005

AbstractWe have investigated the performance of two types of standard flow cell sorter instruments, a System 50 Cytofluorograph and a FACSTar PLUS cell sorter, for the on‐line centromeric index (CI) analysis of human chromosomes. To optimize the results, we improved the detection efficiency for centromeres in two ways. A higher efficiency was obtained first by elongation of the chromosomes and second by introducing a high resolution lens system for laser beam focusing. In the two‐parameter flow karyotype of CI and DNA content of human chromosomes, distinct peaks are produced not only by the larger chromosomes 1‐‐8 and X, but by the smaller nonacrocentric chromosomes 9–12 and 16–20 as well. As the chromosomes 9–12 cannot be distinguished by other flow karyotyping methods, we discriminated and sorted chromosomes 12 and 10 from 9 and 11 to investigate the capacity for the separation of chromosomes in this group. A purity of at least 90% was achieved; in the isolated population the fraction chromosomes 12 was 55%; the remaining 45% were chromosomes 10 (40%) and unidentifiable chromosomes (5%). © 1994 Wiley‐Liss, Inc.
Influence of sampling time on assessment of potential doubling time
Authors: Høyer Morten , Bentzen Søren M. , Salling Lisbeth N. , Overgaard Jens 
Published: June 2005

AbstractPotential doubling time (Tpot), S‐phase transit time (Ts), and labeling index were determined in three experimental rodent tumors by in vivo incorporation of bromodeoxyuridine and flow cytometry. The kinetic parameters were derived from the relative movement method at varying sampling times (time between injection of bromodeoxyuridine and tumor excision). Ts and Tpot were close to the expected (extrapolated) values of the two parameters with sampling time within the range 60–100% of the expected Ts. With short sampling time, Ts and Tpot were considerably over‐ or underestimated. By use of the original method by (Begg et al., Cytometry 6:620–626, 1985), the Tpot came out with a twofold overestimation with a short sampling time. Generally, modified methods for calculation of Ts did not improve the results. When Tpot is measured in human tumors, there is no a priori knowledge of the kinetic parameters. Consequently, the sampling time must be based on the general experience of tumor cell kinetics in the specific tumor type measured. A relatively long sampling time should be aimed for when measuring Tpot in human tumors. With proper sampling time, Tpot assessed by flow cytometry was found to be a precise and reproducible parameter. © 1994 Wiley‐Liss, Inc.
Enhanced assessment of DNA/proliferative index by depletion of tumor infiltrating leukocytes prior to monoclonal antibody gated analysis of tumor cell DNA
Authors: Kenyon Norma S. , Schmittling Robert J. , Siiman Olavi , Burshteyn Alexander , Bolton Wade E. 
Published: June 2005

AbstractFlow cytometry has become an important tool for the analysis of breast tumors, and assessment of S phase fraction and DNA ploidy are potential indicators of tumor aggression. Due to masking or dilution of infrequent tumor cell events, the presence of normal cell types, such as inflammatory cells and fibroblasts, can interfere with accurate DNA analysis of solid tumor samples. MDA‐MB‐175‐VII human breast carcinoma cells, WI‐38 human lung fibroblast cells, and peripheral blood leukocytes were mixed, in varying proportions, in order to represent human breast tumor samples. The cells were subsequently treated with CD45 conjugated magnetic microspheres to deplete tumor infiltrating leukocytes, thus enriching for tumor cells. The tumor cell mixtures were then stained with a pancytokeratin specific monoclonal antibody or with a monoclonal antibody that reacts with breast epithelial membrane antigen (EMA). When used in combination with monoclonal antibody gating, utilization of this bead‐based technology resulted in enhanced precision of DNA analysis. © 1994 Wiley‐Liss, Inc.
Time‐resolved flow cytometry for the measurement of lanthanide chelate fluorescence: I. Concept and theoretical evaluation
Authors: Condrau Marc A. , Schwendener Reto A. , Niederer Peter , Anliker Max 
Published: June 2005

AbstractThe concept of a flow cytometer suited for the time‐resolved measurement of lanthaide chelate luminescence with a decay time on the order of 10 μs to 2 ms is presented and evaluated. The instrument proposed encompasses a continuouswave laser for fluorescence excitation and an optical switch for the elimination of cellular autofluorescence decaying within 1 ns to 1 μs during the luminescence detection period. The slowly decaying fluorescence is to be quantified by a photon‐counting system, whereas light scatter and prompt fluorescence parameters are acquired by a conventional detection system.The detection limit of the method, in terms of the smallest detectable number of fluorescing chelates per cell, is examined. It was found to be nearly 30,000 complexes of a europium chelate with a decay time of 1.6 ms and a quantum efficiency of 17%, independent of fast decaying cellular autofluorescence or prompt dye emission intensity. The probability of cells passing through the instrument without being detected while the laser beam is turned off was estimated, and the implications for cell throughput and sorting performance of the instrument were assessed. At typical fluorescence detection intervals of 500 μs to 1 ms, cell flow rates of 100–200 particles per second lead to detection probabilities of more than 90% and sorting purities comparable to those found in conventional fluorescence‐activated cell sorting. © 1994 Wiley‐Liss, Inc.
Preservation of B‐cell‐associated surface antigens by chemical fixation
Authors: Caldwell Charles W. 
Published: June 2005

AbstractUsual methods of chemical fixation preclude examination of cells with most monoclonal antibodies due to alteration or destruction of the surface antigen itself. A method of chemical stabilization and preservation of human B‐cell‐associated surface antigens is described which facilitates retrospective flow cytometric analysis. This method involves pretreatment of the cells with protease enzyme inhibitors, followed by chemical crosslinking of surface proteins with 2% formalin, and finally blockade of non‐specific reactive groups with excess glycine. Once prepared, the expression of pertinent cellular antigens is stable on the cell surface for as long as 4 years. Such methodology could conceivably be used for preparation of cells for longitudinal quality control of monoclonal antibodies or archival storage of patient specimens for retrospective flow cytometric analysis. © 1994 Wiley‐Liss, Inc.
Detection of terminal transferase in acute myeloid leukemia by flow cytometry
Authors: Paietta Elisabeth , Meenan Bernadette , Heavey Claire , Thomas Dylan 
Published: June 2005

AbstractThe purpose of this study was twofold: (1) to develop an optimized, reliable method for the flow cytometric analysis of the intranuclear DNA polymerase, terminal deoxynucleotidyl transferase (TdT) in acute myeloid leukemia, and (2) to establish the usefulness of a novel, fluorescein‐isothiocyanate conjugated monoclonal anti‐TdT antibody (HT‐6) in double‐fluorescence staining for surface antigens in the characterization of leukemic cells. Inclusion of an aldehyde blocking buffer in the staining protocol reduced background fluorescence sufficiently to allow for the detection of the low‐level fluorescent TdT+ myeloblasts. When admixed to normal peripheral blood mononuclear cells, 0.4–0.5% of HLA‐DR+ or myeloid surface antigen+, TdT+ double‐stained myeloblasts could be reliably detected above background levels. Flow cytometric TdT measurements using the HT‐6 antibody in 55 patients with TdT+ acute lymphocytic or myelocytic leukemia or blast crisis of chronic myelogenous leukemia were equal or superior to the results obtained with a mixture of monoclonal anti‐TdT antibodies (anti‐HTDT‐Mix) and comparable to those obtained by the conventional slide method employing polyclonal rabbit anti‐human TdT antiserum. This flow cytometric TdT determination in combination with surface antigen staining using a novel anti‐TdT monoclonal antibody (HT‐6) allows for the recognition of minimal leukemic blast cells during clinical remission in acute myeloid leukemia. © 1994 Wiley‐Liss, Inc.
Counting, measuring, and mapping in fish‐labelled cells: Sample size considerations and implications for automation
Authors: Carothers Andrew D. 
Published: June 2005

AbstractStatistical models are used to investigate the need for automation in several potential areas of application of FISH‐la‐belling techniques, including perinatal and tumour cytogenetics, genetic toxicology, and gene mapping. Predictions of the models, based on current estimates of likely error rates for spot‐counting and measuring, suggest that a fully automated system is a realistic prospect for detecting full or high‐level mosaic trisomies and that interactive systems have the potential to reduce substantially the human workload required to detect residual malignant disease or radiation‐induced chromosome aberrations. There appear to be no foreseeable limits to the requirements for speed and accuracy in such systems, since there is effectively no lower limit to the level of relevant biological detail that can be investigated with these techniques. © 1994 Wiley‐Liss, Inc.
Interactive computer‐assisted analysis of chromosome 1 colocalization with nucleoli
Authors: Léger Isabelle , Guillaud Martial , Krief Bruno , Brugal Gérard 
Published: June 2005

AbstractThe applications of DNA cloning and fluorescent in situ hybridization (FISH) techniques have strengthened the hypothesis of an ordered chromatin structure in interphase nuclei, strongly suspected to vary with functional state. The nonrandom distribution of the centromeres and their dynamic rearrangement during the cell cycle have been well documented. A close proximity of specific centromeres to nucleoli has also been reported, but the functional meaning of this association is still unknown. In order to investigate whether the chromosome 1 centromere region to nucleolus association depends on the cell cycle and chromosome status, we combined FISH of probes specific for the 1q12 region with Ki‐67 nucleolar antigen fluorescent immunocytochemical (FICC) detection on the MCF‐7 human breast cancer cell line and on the MRC‐5 normal fibroblastic cell line. Both FISH and FICC signals were interactively localized in a one‐step fluorescent microscopic observation and further analyzed using the Highly Optimized Microscope Environment (HOME) graphics microscope workstation, which provided computerized interactive marking of 1q12 to nucleolus associations (1q12‐nu) at the individual nucleus and nucleolus levels. This study confirms that centromeric regions, other than those adjacent to the major ribosomal cistrons, contribute to the perinucleolar chromatin and demonstrate that, during the cell cycle, the heterochromatic band 1q12 is dynamically rearranged with regard to both the nuclear volume and the nucleoli. A relationship between the association of the chromosome 1 pericentromeric region with nucleoli and the nucleolar transcriptional activity is also strongly suggested. © 1994 Wiley‐Liss, Inc.
Fast kinetic measurements and on‐line dilution by flow injection cytometry
Authors: Lindberg Walter , Scampavia Louis D. , Ruzicka Jaromir , Christian Gary D. 
Published: June 2005

AbstractAn improved flow injection cytometry (FIC) system suitable for fast cellular kinetic measurements and on‐line dilution is described. The instrument allows for measurements from 1.2 s (± 0.05 s) after the initiation of mixing, up to any time thereafter. Crucial factors in determining fast kinetic measurements, such as the displacement volume for sample introduction, rate of transport to cytometric interrogation point, and the mixing speed are evaluated and discussed. By varying the volume aspirated, this instrument can facilitate the dilution of cells and/or reagent over a range of one order of magnitude. The fast kinetic and on‐line dilution capabilities were demonstrated by on‐line staining of DNA in trout erythrocytes with 4′, 6‐diamidino‐2‐phenylindole (DAPI). The utility of the instrument for measurement of enzyme kinetics was illustrated using human lymphocytes, measuring the glutathione S‐transferase (GST) catalyzed reaction between monochlorobimane (MCB) and glutathione (GSH). © 1994 Wiley‐Liss, Inc.
Reversing the effect of formalin on the binding of propidium iodide to DNA
Authors: Overton W. Roy , McCoy J. Philip 
Published: June 2005

AbstractFormalin, an excellent preservative of cellular morphology, is a commonly used fixative for tissue specimens in hospital pathology laboratories. This preserved material is a potential source of tissue for diagnostic and retrospective research studies on DNA using flow cytometry. Unfortunately, formalin interferes with the binding of propidium iodide (PI) and other fluorescent dyes to DNA, thus altering the measurement of DNA content by flow cytometry or image analysis. This interference has been attributed to the cross‐linking of histones by formalin. Since formalin alters the measurement of DNA content in formalin‐fixed and formalin‐fixed, paraffin‐embedded tissues, this study was designed to explore the use of various physicochemical methods to reverse the effect of the formalin on the binding of PI to DNA. This study demonstrates that resuspending formalin‐fixed cells in PBS and heating them at 75°C for at least 1 h prior to staining with PI restores the staining of the DNA to approximately the same fluorescence intensity as that of fresh tissue. © 1994 Wiley‐Liss, Inc.
Pattern sorting: A computer‐controlled multidimensional sorting method using k‐d trees
Authors: Bigos M. , Parks D. R. , Moore W. A. , Herzenberg L. A. , Herzenberg L. A. 
Published: June 2005

AbstractMultidimensional binary trees provide a memory efficient and general method for computing sorting decisions in real time for a flow cytometer. Their fundamental advantage over conventional lookup table sorting techniques is that sort criteria in the full N‐dimensional data space which cannot be described by projections onto two‐dimensional parameter planes can be effectively implemented. This becomes particularly relevant when multidimensional analysis methods such as principal components or clustering are employed. We describe a prototype implementation of this method and point out other possible implementations. © 1994 Wiley‐Liss, Inc.
A flow cytometric method using Hoechst 33342 and propidium iodide for simultaneous cell cycle analysis and apoptosis determination in unfixed cells
Authors: Belloc Francis , Dumain Patrice , Boisseau Michel R. , Jalloustre Claudine , Reiffers Josy , Bernard Philippe , Lacombe Francis 
Published: June 2005

AbstractA flow cytometric method to detect apoptotic cells is described. This method is based on the detection of differences in chromatin condensation with Hoechst 33342 as a probe and the detection of dead cells with propidium iodide as a probe for membrane damage. By this method it was possible to detect, in the same sample and at the same time, intact cells, cells undergoing apoptosis, and dead cells resulting from apoptotic and/or necrotic processes. The method was successfully applied to the detection of apoptotic cells in two human cell models: cultured polymorphonuclear cells and the U937 cell line treated with antitumoral drugs. Staining specificity for apoptotic cells was controlled by cell sorting of the presumed apoptotic population, followed by morphologic examination or DNA analysis of the sorted populations. The usefulness of such a method is discussed in terms of applications in the analysis of heterogeneous clinical samples, populations with low DNA degradation during apoptosis, and cell cycle position of the apoptotic cells. © 1994 Wiley‐Liss, Inc.
The macro flow planktometer: A new device for volume and fluorescence analysis of macro plankton including triggered video imaging in flow
Authors: Hüller Reinhold , Glossner Ewald , Schaub Stefan , Weingärtner Johann , Kachel Volker 
Published: June 2005

AbstractA newly developed macro flow planktometer is described for measuring plankton organisms with a size of 100–2,000 μm by the flow principle in time‐saving and noncontact fashion. Based on the method of changes in electric resistance (Coulter principle), the device allows determination of the body volume of living organisms in their natural medium (fresh or seawater) at a measuring rate of up to 50 organisms per second. Simultaneous, laser‐excited fluorescence measurement permits quantitative detection of fluorescent substances within the organisms. Controlled by the multiparametric measurement, video images of organisms of particular interest can be taken in the flow chamber using a stroboscopic imaging system. Valuable morphological information on the material under test is thus available in addition to the analytical measurement. Data acquisition and imaging, the total system control, and the fast data evaluation are performed with an IBM‐AT compatible computer using an extensive software package. The easy‐to‐handle desktop unit can be used both in land laboratories and on ships. The modular structure of the system permits any desired combination of individual components to adapt it to various requirements. The operability of this new measuring system is demonstrated by several applications. © 1994 Wiley‐Liss, Inc.
A disposable‐chamber temperature‐regulation system for the study of intracellular calcium levels in single live T cells using fluorescence digital‐imaging microscopy
Authors: Boltz R. C. “Dutch” , Sirotina Anna , Blake Tom , Kath Gary , Uhrig Brian , McKeel John , Quinn Colleen 
Published: June 2005

AbstractUtilizing flow cytometry, we previously demonstrated that the potassium channel blocker margatoxin (MgTX) inhibits the[Ca2+]i transient involved in T‐cell activation. We wished to extend these studies to single‐cell transients using florescence digital‐imaging microscopy (DIM). However, the most currently available temperature‐regulation chambers reuse part or all of the apparatus and introduce compounds via perfusion. Thus, these apparatuses are not suitable for studies involving compounds that are particularly sticky. We have designed a dual‐temperature regulation system that will maintain Nunc, eight‐well, coverglass‐bottom, disposable chambers, and three disposable addition pipets at 37°C for physiological studies on an inverted digital‐imaging microscope. We have demonstrated that calcium transients of human T lymphocytes can be initiated and monitored reproducibly during the addition of three distinct chemical species. The DIM results correlate with flow cytometry measurements in the number of responding cells and the heterogeneity of the response in both control and MgTX‐inhibited cultures. Additionally, DIM revealed that the [Ca2+]i transient is more rapid than the flow‐cytometric measurement indicated. The correlation between flow cytometry and DIM permits the interpretation of studies on the regulation of T‐cell activation. © 1994 Wiley‐Liss, Inc.
Textural differences between AA and SS blood specimens as detected by image analysis
Authors: Robinson Roy D. , Benjamin Lennette J. , Cosgriff Janice M. , Cox Christopher , Lapets Oleg P. , Rowley Peter T. , Yatco Edward , Wheeless Leon L. 
Published: June 2005

AbstractHigh‐resolution image analysis was employed in the analysis of round (discoid) erythrocytes from hematologically normal (AA) individuals, AA individuals with nonspecific anemia, individuals with sickle cell trait (AS), individuals with SC disease (SC), and individuals with sickle cell anemia (SS). The shape feature Form Factor (4πArea/Perimeter2) was used to select round cells and to exclude sickle and other abnormal cells. Textural features extracted from round cells of SS and SC patients were found to differ from those derived from cells of normal andanemic AA individuals. Two textural features, Standard Deviation of Run Length Matrix Counts and Rotation Moment of the Cooccurrence Matrix, discriminated between patient mean values from AA samples and those from SS samples. The ability of textural features to separate round cells into classes based on genotype suggests that high resolution image analysis may be an effective tool in the study and monitoring of sickle cell disease. © 1994 Wiley‐Liss, Inc.
TO‐PRO‐3 iodide: A novel HeNe laser‐excitable DNA stain as an alternative for propidium iodide in multiparameter flow cytometry
Authors: Van Hooijdonk Candida A. E. M. , Glade Conrad P. , Van Erp Piet E. J. 
Published: June 2005

AbstractA new red emitting fluorophore, TO‐PRO‐3 iodide (TP3), which is best excited by an HeNe laser (633 nm), has been compared with propidium iodide (PI) for measuring relative DNA content. TP3, which has a peak absorbance at 642 nm and emission at 661 nm, has been tested on peripheral blood lymphocytes (PBL) and keratinocytes in a two‐laser system. As an example, we present a three‐color flow cytometric application utilizing TP3 in combination with fluorescein‐isothiocyanate (FITC) and phycoerythrin (PE) conjugated to monoclonal antibodies in this paper.A subequilibrium concentration of 1 μM TP3, most preferably used in combination with RNase treatment, proved to be a powerful alternative for DNA amount determination. In human‐ and mouse‐Balb/MK‐keratinocyte populations with different S‐phase fractions, PI and TP3 showed a good correlation. Finally, in the triple labelling experiment we clearly demonstrated that TP3 is readily applied to the analysis of binding of two antibodies and relative DNA content simultaneously. © 1994 Wiley‐Liss, Inc.