bluuebunny/crossref_metadata_2025_split huggingface dataset:
Flow cytometric detection of mitotic cells using the bromodeoxyuridine/DNA technique in combination with 90° and forward scatter measurements
Authors: Nüsse Michael , Jülch Markus , Geido Elio , Bruno Silvia , Vinci Angela Di , Giaretti Walter , Ruoss Kurt 
Published: August 2005

AbstractMitotic cells could be well discriminated from the cells in the G1‐, S‐ and G2‐phases of the cell cycle using pulse labeling of S‐phase cells with bromodeoxyuridine (BrdUrd) and staining of the cells for incorporated BrdUrd and total DNA content. Unlabeled G2‐ and M‐phase cells could be measured as two separate peaks according to propidium iodide fluorescence. M‐phase cells showed lower propidium iodide fluorescence emission compared to G2‐phase cells. The fluorescence difference of M‐ and G2‐phase cells was caused by the different thermal denaturation of their DNA. Best separation of M‐and G2‐phase cells was obtained after 30–50 min heat treatment at 95°C. Mitotic index could be measured if no unlabeled S‐phase cells were present in the cell culture. With additional measurements of 90° scatter and/or forward scatter signals, mitotic cells could be clearly discriminated from both unlabeled G2‐ and S‐phase cells. The correct discrimination (about 99%) of mitotic cells from interphase cells was verified by visual analysis of the nuclear morphology after selective sorting. Unlabeled and labeled mitotic cells could be observed as pulselabeled cells progressed through the cell cycle. We conclude that this modified BrdUrd/DNA technique using prolonged thermal denaturation and the simultaneous measurement of scatter signals may offer additional information especially in the presence of BrdUrdunlabeled S‐phase cells.
Automated detection and recognition of live cells in tissue culture using image cytometry
Authors: Spadinger Ingrid , Poon Steven S. S. , Palcic Branko 
Published: August 2005

AbstractAn automated image cytometry device, the Cell Analyzer, was used to locate live V79 cells plated at low densities in a tissue culture flask. Cells and other objects were detected by moving the flask in steps across a linear solid‐state image sensor. The step size was selected to be small enough to allow detection of all the cells in the area being scanned but sufficiently large so that most cells would be detected on only one image line. To distinguish cells from other detected objects, a recognition algorithm utilizing 18 characteristic cell signal features was developed. The algorithm first tests whether a set of feature values falls within specified upper and lower bounds, and then applies a linear discriminant function to the remaining data to further discriminate cells from debris. False‐positive errors of 5% or less were achieved with this method, whereas 15–35% of cells were misclassified as debris.
Central values and variation of measured nuclear DNA content in imprints of normal tissues determined by image analysis
Authors: Taylor Suzanne R. , Titus‐Ernstoff Linda , Stitely Suzette 
Published: August 2005

AbstractA total of 109 slides derived from 37 normal tissues were analyzed for nuclear DNA content using the Cell Analysis Systems (CAS) model 100 image analysis system with the Quantitative DNA Analysis (QDA) software module in order to determine the central values of DNA content and define normal limits. Analysis of the 109 slides revealed an overall mean measured DNA index of 0.997 with a standard deviation of 0.04. Analysis of replicate samples was essentially constant. There were no differences between samples obtained from autopsy and surgical specimens. Small variations were detected among stain batches, individual patients, and tissue types. These results indicate a high degree of accuracy and reproducibility of DNA content determinations using this system.
Resolving leukocytes using axial light loss
Authors: Stewart Carleton C. , Stewart Sigrid J. , Habbersett Robert C. 
Published: August 2005

AbstractAxial light loss (ALL) is the measurement of the total light lost from the laser beam at 0 degrees when a particle passes through the beam. Used in combination with the monoclonal antibody CD45, ALL can effectively resolve lymphocytes, monocytes, granulocytes, and dead cells in viable or fixed preparations of human peripheral blood. A bivariate display of ALL vs. CD45 clearly resolves all granulocytes from lymphocytes; although degranulated granulocytes cannot be resolved with forward‐angle and right‐angle light scatter, they are clearly resolved in right‐angle scatter vs. CD45. A blood differential can be performed, with a single laser flow cytometer and three colors of fluorescence, when ALL is combined with fluoresceinated CD45 to resolve leukocytes, phycoerythrin‐labeled NKH1 to resolve natural killer cells, and biotinylated CD3 in combination with DuoCHROME, the phycoerythrin/Texas red conjugate fluorochrome from Becton Dickinson, to resolve T‐cells. B‐cells are the only cells negative for both phycoerythrin and Texas red. When PE CD4 is included, the CD3+ CD4+ T‐cell subset is resolved from the CD3+ CD4− subset comprising mainly the CD3+ CD8+ T‐cell subset. The addition of propidium iodide is unnecessary since ALL clearly resolves dead cells in a viable preparation of human peripheral blood. Furthermore, since ALL resolves these cells even after fixation in paraformal‐dehyde, all samples can be fixed prior to analysis, thereby minimizing the potential biohazard risk.
Flow cytometric analysis of mature adipocytes
Authors: Bernstein R. L. , Hyun William C. , Davis Jonathan H. , Fulwyler Mack J. , Pershadsingh Harrihar A. 
Published: August 2005

AbstractFlow cytometry is an excellent method for studying the physiological function in adipocytes because their response to hormones, especially insulin, varies with cell maturity and therefore size. Adipocytes present a unique technical challenge. A freshly prepared adipocyte suspension contains cells and fat droplets ranging from 10 to >120 μm in diameter. Stored fat occupies 90–98% of the cell volume, making it difficult to distinguish cells from fat droplets. Other difficulties include buoyancy, large size, fragility, and tendency to aggregate and clog the sample tube and nozzle.These obstacles were overcome by (1) maintaining the sample, sample line, sheath fluid, reservoir, and nozzle assembly at 37°C; (2) using a 200 μm diameter orifice; (3) using a short, 300 μm inside diameter Teflon sample delivery line; (4) injecting the sample at constant flow rate into the sheath fluid at low pressure; and (5) using the pH‐sensitive vital stain, biscarboxyethylcarboxyfluorescein (BCECF) to distinguish cells from fat droplets.Stained cells are brightly fluorescent when excited at 488 nm. Because fat droplets do not fluoresce, they can be distinguished from fat cells by gating on the BCECF emission. The cytosolic pH of intact, viable, mature adipocytes was derived from the ratio of the fluorescent emission intensities at 520 and 620 nm and was estimated to be 7.2. Unlike BCECF, several other useful fluorescent probes of cell function, e.g., the intracellular calcium indicator, indo‐1, label both fat cells and fat droplets. Using a dual‐excitation‐laser flow cytometer and by gating on the BCECF emission in an adipocyte suspension labeled with both BCECF and indo‐1, one can exclusively monitor cell‐associated indo‐1 fluorescence in order to assess unambiguously intracellular calcium concentration in viable, mature adipocytes.
Optical plankton analyser: A flow cytometer for plankton analysis, I: Design considerations
Authors: Peeters J. C. H. , Dubelaar G. B. J. , Ringelberg J. , Visser J. W. M. 
Published: August 2005

AbstractThe design criteria for a flow cytometer (FCM) for the analysis of field samples of phytoplankton are described. The criteria are based on the occurrence of a wide variety of particle sizes in field samples, normally at low concentrations. The instrument should be able to analyse cells and colonies from 0.5 to 500 μm diameter and of over 2,000 μm length. A minimum flow rate of 4 μl · s−1 was calculated from natural plankton concentrations. Commercially available FCMs are not suited to measure this range of sizes at this rate. Further limitations of standard FCMs are uneven illumination or incomplete processing of long signals. In addition, long filamentous colonies can break into small fragments caused by too high acceleration in the standard flow cuvette. Recognition of these limitations is of importance for the flow cytometry of phytoplankton.The new design was developed to avoid these limitations. A dynamic range 5 to 6 decades could be accomplished by a combination of logarithmic amplifiers, a slit‐shaped focal spot, and a pulse integration system that can process long pulses. Multilaser capability to identify different phytoplankton species, a low fluid shear cuvette, and a trigger gate‐extension for inhomogeneously fluorescent algal filaments were included in the design.
Optical plankton analyser: A flow cytometer for plankton analysis, II: Specifications
Authors: Dubelaar George B. J. , Groenewegen Ad C. , Stokdijk Willem , Van Den Engh G. J. , Visser Jan W. M. 
Published: August 2005

AbstractAn analysing flow cytometer, the optical plankton analyser (OPA), is presented. The instrument is designed for phytoplankton anaysis, having a sensitivity comparable with commercially available flow cytometers, but a significantly extended particle size range. Particles of 500 μm in width and over 1,000 μm in length can be analysed. Sample flow rates of up to 55 μl/s can be used. Also, the dynamic range of the instrument is significantly increased for particles larger than about 5 μm. The optics, hydraulics, and electronics of the instrument are described, including the best form for a low fluid shear cuvette. The new pulse quantification technique we call digital integration is presented. This technique is essential for the instrument to handle both short and very long particles with a large dynamic range. Test measurements demonstrating particle size range and dynamic range are presented. Dynamic ranges of 10,000 and 100,000 were typically observed, measuring field samples with Microcystis aeruginosa colonies, whereas one sample showed a dynamic range of 106. A simple method for interpretation of time of flight (TOF) data in terms of particle morphology is presented. The specifications of the instrument are given.
Use of a neural net computer system for analysis of flow cytometric data of phytoplankton populations
Authors: Frankel Donald S. , Olson Robert J. , Frankel Sheila L. , Chisholm Sallie W. 
Published: August 2005

AbstractFlow cytometry has been used over the past 5 years to begin detailed exploration of the distribution and abundance of picoplankton in the oceans. Light scattering and fluorescence measurements on individual plankton cells in seawater samples allow construction of population signatures from size and pigment characteristics. The use of “list mode” data has made these studies possible, but on‐shore analysis of copious data does not permit on‐site reexamination of important or unexpected observations, and overall effort is greatly handicapped by data analysis time.Here we describe the application of neural net computer technology to the analysis of flow cytometry data. Although the data used in this study are from oceanographic research, the results are general and should be directly applicable to flow cytometry data of any sort.Neural net computers are ideally suited to perform the pattern recognition required for the quantitative analysis of flow cytometry data. Rather than being programmed to perform analysis, the neural net computer is “taught” how to analyze the cell populations by presenting examples of inputs and correct results. Once the system is “trained,” similar data sets can be analyzed rapidly and objectively, minimizing the need for laborious user interaction. The neural network described here offers the advantages of (1) adaptability to changing conditions and (2) potential real‐time analysis. High accuracy and processing speed near that required for real‐time classification have been achieved in a software simulation of the neural network on a Macintosh™ SE personal computer.
Algorithm to estimate cell biovolume using image analyzed microscopy
Authors: Sieracki Michael E. , Viles Charles L. , Webb Kenneth L. 
Published: August 2005

AbstractThis paper describes an algorithm for calculating the biovolume of cells with simple shapes, such as bacteria, flagellates, and simple ciliates, from a 2‐dimensional digital image. The method can be adapted to any image analysis system which allows access to the binary cell image—(i.e.), the pixels, or (x, y) points, composing the cell. The cell image is rotated to a standard orientation (horizontal), and a solid of revolution is calculated by digital integration. Verification and a critical assessment of the method are presented. The algorithm accounts for irregularities in cell shape that conventional methods based on length, width, and geometrical formulas do not.
Kinetics of bacterial processes in natural aquatic systems based on biomass as determined by high‐resolution flow cytometry
Authors: Button D. K. , Robertson B. R. 
Published: August 2005

AbstractThe two primary kinetic constants for describing the concentration dependency of nutrient uptake by microorganisms are shown to be maximal rate of substrate uptake and, rather than the Michaelis constant for transport, specific affinity. Of the two, the specific affinity is more important for describing natural aquatic microbial processes because it can be used independently at small substrate concentrations. Flow cytometry was used to evaluate specific affinities in natural populations of aquatic bacteria because it gives a convenient measure of biomass, which is an essential measurement in the specific‐affinity approach to microbial kinetics.Total biomass, biomass in various filter fractions, and the specific affinity of the bacteria in each fraction were determined in samples from a near‐arctic lake. The partial growth rate of the pelagic bacteria from the 25 μg/liter of dissolved amino acids present (growth rate from the amino acid fraction alone) was determined to be 0.78 per day. By measuring activity in screened and whole‐system populations, the biomass of the bacteria associated with particles was computed to be 427 μg/liter.
Shipboard analytical flow cytometry of oceanic ultraphytoplankton
Authors: Li William K. W. 
Published: August 2005

AbstractShipboard analysis of marine ultraphytoplankton by flow cytometry is a powerful method to classify these cells according to in vivo fluorescence characteristics and size. At present, this ataxonomic‐allometric approach allows recognition of phycoerythrin‐containing cyanobacteria, cryptomonads, very small red‐fluorescing cells (presumably prochlorophytes), and eukaryotic algae of various sizes in many open ocean samples. The speed at which flow cytometric analysis can be performed on freshly collected samples permits a high degree of sampling resolution in both space and time. A flow cytometric view is presented of the vertical distribution of ultraphytoplankton at various sites in the north Atlantic and of experiments wherein phytoplankton were incubated in an artificial light gradient and under simulated in situ conditions.
Variance within homogeneous phytoplankton populations, I: Theoretical framework for interpreting histograms
Authors: Campbell J. W. , Yentsch C. M. 
Published: August 2005

AbstractA framework is presented for interpreting frequency distributions of volume or fluorescence as measured by a flow cytometer on homogeneous phytoplankton populations. The framework, based on both laboratory experience and theoretical concepts, is illustrated with the use of a simulation model. Asynchronous, synchronous, and phased populations were simulated, with constant and variable growth patterns over the cell cycle. Though simulations produced a wide variety of histogram shapes, including multimodal distributions, the primary difference between asynchronous and synchronous/phased distributions lies in their temporal variation. Histograms that are constant in time indicate asynchronous populations; when populations are not asynchronous, their histogram shapes vary with a periodicity on the same time scale as the cell cycle. A probability density function for the case of asynchronous populations with a constant growth rate is derived. When fitted to simulated histograms this two‐parameter density function yields estimates of the two parameters: mean and variance of cell volume (or mass) at age 0.
Carbon dynamics of logarithmetic and stationary phase phytoplankton as determined by track autoradiography
Authors: Knoechel R. , Quinn E. M. 
Published: August 2005

AbstractTrack autoradiographic analysis of photosynthetic radiocarbon incorporation at the cellular level indicated that the carbon uptake rate and carbon pool size of exponentially growing (log phase) Scenedesmus cells was threefold that of stationary phase cells, while carbon turnover rates were similar. Carbon fixation was uncoupled from growth and cell division in the stationary phase cells, which were larger and contained less chlorophyll per unit volume than log phase cells. Changes in the temporal pattern of isotope incorporation were evident at the cell level prior to the cessation of division and transition to stationary phase, while bulk carbon fixation responded only the second day after cell division ceased. The carbon uptake patterns of a marine nanoplankter from a nutrient‐enriched natural sample resembled that of log phase cells while the control population pattern resembled that of stationary cells. The physical, biochemical, and metabolic differences between log and stationary phase cells are potentially measurable by flow cytometry procedures currently in use and under development. The use of flow cytometry to sort cell types for analysis by track auto‐radiography and subsequent correlation of metabolic characteristics with flow cytometry signatures is a feasible means of investigating the heterogeneity of phytoplankton metabolic state in the marine environment.
Discrimination of eukaryotic phytoplankton cell types from light scatter and autofluorescence properties measured by flow cytometry
Authors: Olson R. J. , Zettler E. R. , Anderson O. K. 
Published: August 2005

AbstractFlow cytometric methods for recognizing several groups of eukaryotic marine phytoplankton were tested using 26 laboratory cultures. Each culture was divided into three aliquots, and these samples were analyzed for (1) Coulter volume; (2) light scatter (magnitude and polarization properties of forward scattered light and magnitude of right‐angle scattered light) and autofluorescence emission (phycoerythrin and chlorophyll); and (3) autofluorescence excitation (by 488 nm and 515 nm light).Three kinds of cells could be easily distinguished from others in the culture collection: (1) The two cryptophytes and the rhodophyte had high phycoerythrin/chlorophyll ratios; (2) the two coccolithophores depolarized forward scattered light; and (3) the two pennate diatoms scattered only a relatively small amount of light in the forward direction compared with that at right angles. Mean chlorophyll fluorescence excited by blue light relative to that excited by green light was highest in the four chlorophytes, but there was overlap between some of these and some other kinds of cells. Unresolved cell types included centric diatoms, dinoflagellates, and naked coccolithophores. Forward light scatter and Coulter volume were closely related (except for the pennate diatoms) over a range of about 0.01 to 30 pL (equivalent spherical diameter about 3 to 40 μm), according to a logarithmic function.
Using phytoplankton and flow cytometry to analyze grazing by marine organisms
Authors: Cucci Terry L. , Shumway Sandra E. , Brown Wendy S. , Newell Carter R. 
Published: August 2005

AbstractPhytoplankton can, through their auto‐fluorescent characteristics, be thought of as tracer particles in much the same way as fluorescent microspheres when used in particle uptake experiments. Flow cytometric techniques can be used to differentiate phytoplankton from other suspended particles by the two primary autofluorescing photosynthetic pigments, chlorophyll and phycoerythrin. Based on these characteristics, phytoplankton assemblages have been used to assess grazing rates, particle selectivity, and endocytotic abilities in various marine species, from single‐celled organisms to higher invertebrates.
Reference standards for flow cytometry and application in comparative studies of nuclear DNA content
Authors: Tiersch Terrence R. , Chandler Robert W. , Wachtel Stephen S. , Elias Sherman 
Published: August 2005

AbstractNuclear DNA mass in cells from a reference species can be used to obtain high‐resolution estimates of DNA mass from a target species. In our study of DNA mass in cells from 45 selected species, representing each of the major vertebrate classes, we have obtained values of from 1.5 to 110.0 pg of DNA. Because values in or near this range would be expected in the study of nuclear DNA mass in vertebrates and other organisms, the species in this report can provide a useful catalogue of references for comparative studies of DNA.
Different opinions on classification of DNA histograms produced from paraffin‐embedded tissue
Authors: Joensuu Heikki , Kallioniemi Olli‐Pekka 
Published: August 2005

AbstractFlow cytometric DNA ploidy determination has been regarded as an objective prognostic parameter in several types of human cancer. To test whether DNA histograms are similarly interpreted, a series of flow cytometric DNA histograms was posted to six investigators working in the field for independent classification. The histograms were produced from paraffin‐embedded adrenal adenomas or non‐neoplastic tissue and had several different patterns. Only 44% of the histograms were similarly classified by all investigators, and 85% by five of the six participants, when DNA ploidy was evaluated. Different criteria for tetraploidy existed, and also some uncertainty in classifying peridiploid and small aneuploid peaks. It is concluded that lack of consensus on histogram classification may result in widely varying percentages of DNA aneuploid tumors found even if the data are similar. Until general agreement is reached on the definition of DNA aneuploidy and its subclasses, classification of DNA histograms is variable and subjective.
Thermal denaturation of DNA for immunochemical staining of incorporated bromodeoxyuridine (BrdUrd): Critical factors that affect the amount of fluorescence and the shape of BrdUrd/DNA histogram
Authors: Hoy C. A. , Seamer L. C. , Schimke R. T. 
Published: August 2005

AbstractSignificant inter‐ and intraexperimental variations of the relative antibromodeoxyuridine fluorescence were found during measurement of DNA synthesis rates using flow cytometric analysis of 5‐bromodeoxyuridine (BrdUrd)‐labeled cells with an anti‐BrdUrd antibody. Fluctuations in other endpoints associated with levels of denaturation (integrity of DNA and cell size) were also observed to vary widely among samples that were otherwise thought to have been treated identically. Therefore, the denaturation step has been carefully re‐examined, and several critical factors were identified that influence the denaturation and subsequent binding of the anti‐BrdUrd to the labeled DNA. These factors include cell density, volume of water, and pH of the sample during heating. Appropriate adjustments are now included in the protocol, resulting in more consistent anti‐BrdUrd measurements in the face of routine (and sometimes necessary) experimental variations.