bluuebunny/crossref_metadata_2025_split huggingface dataset:
Pulse modulation of the excitation light source boosts the sensitivity of an arc lamp‐based flow cytometer
Authors: Steen Harald B. , Sørensen Otto I. 
Published: August 2005

AbstractIt has been found that the type of short arc high‐pressure lamps used in some flow cytometers can be pulsed to reach intensities many times higher than that measured when they are operated on constant power. A 75 W mercury‐xenon lamp was fed 20 m̈s current pulses superimposed on its rated current of 5.4 A. Pulses of 50 A produced a 75‐fold increase of the emission intensity at the excitation wavelength of FITC, which means that the excitation intensity in the flow chamber at this wavelength exceeded 100 mW. At the major emission lines of mercury, i.e., 366, 436, and 546 nm, the increase was about 25‐fold, corresponding to intensities of the order of 300 mW. The light pulses were found to be reproducible to within 2% and the intensity was independent of the pulse frequency up to at least 250 pulses/s. Operated in this mode the lamp produced more than 1 · 108 30 A pulses, which is sufficient to measure some 10,000 typical size samples. The rise time of the light pulses was about 5 m̈s. This was sufficiently short that the leading edge of the light scattering signal from a cell entering the excitation focus of the flow cytometer could be used to trigger the current pulse so that the cells were exposed to the full light pulse intensity while they were still within the focus. Since the sensitivity of flow cytometers is proportional to the square root of the excitation intensity, the resulting increase of the sensitivity should correspond to a lowering of the detection limit for FITC by a factor of about 8. The actual improvement as measured by means of calibrated FITC‐labelled particles was 9.8, thus bringing the FITC sensitivity of the present arc lamp‐based flow cytometer on par with that of the best laser‐based instruments commercially available. © 1993 Wiley‐Liss, Inc.
Phase‐resolved fluorescence lifetime measurements for flow cytometry
Authors: Pinsky Bertram G. , Ladasky John J. , Lakowicz Joseph R. , Berndt Klaus , Hoffman Robert A. 
Published: August 2005

AbstractA flow cytometer capable of measuring fluorescence lifetimes by the phase shift method has been built and evaluated. Under optimal conditions, the resolution of the fluorescence lifetime measurement is shown to be under 200 picoseconds. Pulse intensity variations are normalized using limiting amplifiers and electronic filtering. Normalization of signal intensities provides a lifetime measurement that is independent of fluorescence intensity over at least a 50‐fold (17 dB) range in fluorescence intensity. The fluorescence lifetimes of unbound dye, fluorescent beads, cells stained with ethidium bromide, propidium iodide, and phycoerythrin‐conjugated monoclonal antibodies have been measured. The fluorescence lifetimes measured for these particles are well correlated with lifetime measurements made using a standard fluorimeter. Cells stained with ethidium bromide and propidium iodide at various nucleotide‐to‐dye ratios are shown to exhibit similar behavior to static cuvette measurements. The fluorescence lifetime parameter is also shown to resolve phycoerthyrin fluorescence from propidium iodide fluorescence. © 1993 Wiley‐Liss, Inc.
Immunofluorescent evaluation of DNA repair synthesis using interactive laser cytometry
Authors: Srivastava Vinod , Miller Susan , Busbee David 
Published: August 2005

AbstractAn improved differential fluorescence analysis technique which employs scanning laser cytometry for the detection of two DNA binding fluorochromes was used to quantitate both total DNA and cellular incorporation of the base analogue, bromodeoxyuridine (BrdUrd), into UV‐ or methyl methane sulphonate (MMS)‐treated DNA. In this procedure DNA containing BrdUrd was partially denatured and immunolabeled by binding anti‐BrdUrd monoclonal IgG to incorporated BrdUrd. Anti‐BrdUrd was detected with a secondary fluorochromelabeled polyclonal anti‐IgG. Cells were counterstained with propidium iodide to allow the determination of total DNA. Fluorescence was determined using the Meridian ACAS 570 Scanning Laser Cytometer. The computer‐analyzed images were generated from monodisperse populations of UV‐ or MMS‐treated cells, allowing the evaluation of DNA synthesis associated with excision repair and of total DNA content in single cells. These data were compared with 3H‐thymidine incorporation occurring as a function of excision repair. The analysis of DNA synthesis with this technique is consistent, relatively simple, and rapid with excellent sensitivity and provides a viable method for determining cycling vs. non‐cycling cells, total cellular DNA, and excision repair‐associated DNA synthesis of individual cells within heterogeneous cell populations. © 1993 Wiley‐Liss, Inc.
Shape changes in Escherichia coli B/r A during agar filtration
Authors: Vardi E. , Grover N. B. 
Published: August 2005

AbstractWe have investigated the phenomenon of shape distortion in a sample of 1,552 Escherichia coli B/r A cells in balanced exponential growth, during preparation for electron microscopy by agar filtration. Mixed preparations of bacterial cells and polystyrene latex spheres were shadow cast at low angle and the resulting shadows used to obtain quantitative estimates for the dimensions of the dehydrated cell; these then serve as a basis for a model of its shape in three dimensions. A statistical analysis of the projections of clustered cells and the intervening fissures, in nonshadow‐cast preparations, provides an estimate of the effects of drying. The average width of the dehydrated cell (450 nm) is about 20 nm greater than the diameter of the live bacterium, whereas its length (1,398 nm) is ∼ 40 nm less. © 1993 Wiley‐Liss, Inc.
Flow cytometric clonal excess analysis of peripheral blood, routine handling, and pitfalls in interpretation
Authors: Johnson Anna , Olofsson Tor 
Published: August 2005

AbstractClonal excess (CE) analysis by flow cytometry is a convenient method to detect minimal involvement of peripheral blood and bone marrow by B‐cell lymphoma. The method is based on evaluation of the congruity between κ and λ light chain distributions within a normal B‐cell population. By using the Kolmogorov‐Smirnov method for evaluation of histogram identity, the maximum difference (D value) between distributions is calculated. However, variable adsorption of cytophilic plasma immunoglobulin (Ig) to CD16 positive cells, T‐cell subsets, and the B cells themselves may cause incongruity between the light chain distributions that might simulate or disguise a true clonal excess and thus create major pitfalls in the interpretation of the CE analysis. These phenomena are observed both in normal blood donors and in patients with non‐Hodgkin's lymphoma. In the present report we describe: (1) how freezing of the isolated mononuclear cells before immunostaining effectively removes most of the adsorbed cytophilic Ig, (2) how by exclusion of the CD16 positive population further resolution of the relevant histogram shape is obtained to avoid false interpretations of incongruity, and (3) how adsorption of Ig to B‐cells creates a typical pattern of “reciprocal labeling” that can be mistaken for a clonal excess. Based on our observations we argue against an uncritical use of normal D values for definition of clonal excess and advocate an analytic evaluation of the κ and λ overlay distribution by dual immunofluorescence, either by excluding CD16 positive cells or by including only B cells, to reveal the nature of the different deviations. © 1993 Wiley‐Liss, Inc.
Resolution of fluorescence signals from cells labeled with fluorochromes having different lifetimes by phase‐sensitive flow cytometry
Authors: Steinkamp John A. , Crissman Harry A. 
Published: August 2005

AbstractA flow cytometric method has been developed that uses phase‐sensitive detection to separate signals from simultaneous fluorescence emissions in cells labeled with fluorochromes having different fluorescence decay lifetimes. By CHO cells were stained with propidium iodide (PI) and fluorescein isothiocyanate (FITC). These dyes bind to DNA and protein and the fluorescence lifetimes of the bound dyes are 15.0 and 3.6 ns, respectively. Cells were analyzed as they passed through a modulated (sinusoidal) laser excitation beam. Fluorescence was measured using only a long pass filter to block scattered laser excitation light and a single photomultiplier tube detector. The fluorescence detector output signals were processed by dual‐channel phase‐sensitive detection electronics and the phase‐resolved Pland FITC signals were displayed as frequency distribution histograms and bivariate plots. By shifting the phase of one detector channel reference signal by π/2 + ψ1 degrees and the phase of the other detector channel reference signal by −π/2 + ψ2 degrees, where ψ1 and ψ2 are the phase shifts associated with the PI and FITC lifetimes, the PI and FITC signals were separately resolved at their respective phase‐sensitive detector outputs. This technology is also applicable to suppressing background interferences caused by cellular autofluorescence, unbound/free dye, nonspecific dye binding, and Raman and Rayleigh scattering. © 1993 Wiley‐Liss, Inc.
Flow injection cytometry: A new approach for sample and solution handling in flow cytometry
Authors: Lindberg Walter , Ruzicka Jaromir , Christian Gary D. 
Published: August 2005

AbstractA prototype instrument for sample and solution handling in flow cytometry has been constructed. The system is modular and allows the control of any combination of up to 4 pumps, 2 selection valves, and 2 injection valves. These devices are controlled by a computer using TTL‐logic. The flow injection instrument is interfaced to the flow cytometer via a 6‐port injection valve, thereby facilitating virtually any flow pattern to be implemented without affecting the fluidics of the cytometer. Salient features of the instrument are accurate control of the volume of injected sample and reagent, reproducible timing, and controlled mixing conditions. Results from model experiments of on‐line staining of trout erythrocytes with different concentrations of 4′,6‐diamidino‐2‐phenylindole (DAPI) and propidium iodide (PI) are shown. Possible ways to improve the performance and utility of the instrument are also discussed. © 1993 Wiley‐Liss, Inc.
A comparison of techniques for introducing macromolecules into living cells
Authors: Lee Grace , Delohery Thomas M. , Ronai Zeev , Brandt‐Rauf Paul W. , Pincus Matthew R. , Murphy Randall B. , Weinstein I. Bernard 
Published: August 2005

AbstractFlow cytometry has been used to evaluate several techniques for introducing macromolecules into large numbers of living cells. One technique is cell fusion with red blood cell ghosts loaded with a fluorescent reporter molecule (RBCF). The second technique, termed osmotic lysis of pinosomes (OLP), involves a brief exposure of cells to a hypertonic solution containing the reporter molecule; subsequently, a hypotonic media is added which lyses the pinosomes formed during the hypertonic treatment. A third technique, scrape loading (SL), involves the creation of transient holes in the cell membrane through the application of mechanical forces, which allows for the passage of reporter molecules into cells. A comparison of these techniques is presented here. OLP appears to offer several advantages: It is a simple procedure, virtually all cells are fluorescently labelled, and it is capable of loading larger amounts of material more uniformly into cells while maintaining excellent viability. © 1993 Wiley‐Liss, Inc.
Flow cytometric screening of blood samples for malaria parasites
Authors: van Vianen Philip H. , van Engen Anneloes , Thaithong Sodsri , van der Keur Maarten , Tanke Hans J. , van der Kaay Hugo J. , Mons Barend , Janse Chris J. 
Published: August 2005

AbstractAn automated method for the detection and estimation of malaria parasites in blood samples using flow cytometry is presented. In a single‐step procedure 50 μl of blood sample was collected in 1 ml of lysis solution containing formaldehyde, causing red blood cells to lyse while parasites and white blood cells are preserved. Thus prepared, samples could be transported and remained stored in lysis solution until flow cytometric analysis was performed. The cells were stained for DNA with the fluorescent dye Hoechst 33258 and subsequently analyzed by a FACStar flow cytometer. Parasites and white blood cells were distinguished and counted based on blue Hoechst fluorescence and forward scattering. Since red blood cells were lysed, parasite numbers were given related to the number of white blood cells similar to what is done in microscopic examination of thick blood smears. In dilution experiments with animal and human material, parasite counts by flow cytometry correlated very well with the theoretically calculated numbers (regression coefficients of > 0.94). In human material parasitemias of ∼0.005% were detected. In a pilot Study, 700 samples were collected in Thailand and screened by microscopic examination of thick smears and by flow cytometry; 29 were found positive by combining both methods, 2 were missed by flow cytometry, and 20 were missed by microscopists in the field. After microscopic reexamination in the central laboratory, 15 of these 20 were found positive, 5 remained unconfirmed. © 1993 Wiley‐Liss, Inc.
Nationwide quality control trial on lymphocyte immunophenotyping and flow cytometer performance in Italy
Authors: Brando Bruno , Sommaruga Elisabetta 
Published: August 2005

AbstractThe Italian Society for Cytometry (Gruppo Italiano di Citometria, GIC) promoted this nationwide large‐scale quality control trial on cellular immunophenotyping and flow cytometer performance in 1991. The aim of this independent study was to evaluate instrument performance with calibrated fluorescence microbeads (minimum threshold for FITC, linearity, coefficients of variation and resolution indexes for fluorescence), and to correlate it to the measurement of lyophilized lymphocyte surface immunofluorescence staining with a wide spectrum of antibodies (percentage of positive cells and fluorescence mean intensity).A single send‐out was made to 306 laboratories with 350 instruments throughout Italy. Each kit included anonymous vials containing premixed calibrated microbeads, lyophilized human lymphocytes and small aliquots of conjugated monoclonal antibodies, for both single FITC and double FITC/PE staining. Participants were also asked to use their own anti‐CD4 monoclonal. A valid answer was returned by 209 laboratories with 221 instruments. Gating was not an assay variable. The minimum sensitivity threshold for FITC ranged from 26.6 to 11,293 MESF, with marked instrument heterogeneity as far as the relationship between FITC threshold and coefficients of variation for fluorescence was concerned. A low FITC threshold also correlated with a good resolution index for low intensity stainings. No performance comparisons among instrument brands and models were made. The lyophilized lymphocyte analysis showed an overall frequency of outliers ranging from 3.2% to 19.7%, with a maximum for CD2 FITC (19.7%) and CD3 + HLA−DR+ (12.1%). A strongly negative relationship was evident between the FITC threshold and the number of CD2+ cells, whereas the sensitivity threshold had virtually no effects on the measured level of higher antigen density markers like CD4 and many others. Absolute fluorescence intensity measurements of CD4+ and CD19+ were also made. This study design proved valid and suitable for a large‐scale evaluation of both instrument performance and cytometer operators' skill. A number of practical problems were identified among participants, thus stressing the need for more effective educational programmes and technical guidelines. © 1993 Wiley‐Liss, Inc.
Flow cytometric estimation of poly(A) + RNA by fluorescent in situ hybridization
Authors: Belloc Francis , Lacombe Francis , Dumain Patrice , Mergny Jean Louis , Lopez Francis , Bernard Philippe , Reiffers Josy , Boisseau Michel R. 
Published: August 2005

AbstractA method for the detection of poly(A) + RNA in cell suspensions by in situ hybridization and flow cytometry is described. The hybridizing properties of oligothymidylate o(dT) was well preserved after coupling to fluorescein. This fluorescent oligonucleotide was used as a probe to determine the poly(A) + RNA content of fixed HL60 leukemic cells by flow cytometry. Labeling was considerably reduced by treating the cells with RNAse, and by competitive hybridization with either poly(U) or free poly(A). Labeling was also decreased in a time‐dependent fashion by incubating the cells with actinomycin D prior to fixation. This method represents an improvement on the methods measuring total RNA and could be of value in investigations on the effect of drugs on RNA metabolism. © 1993 Wiley‐Liss, Inc.
Identification of a marker chromosome in a rat ascitic tumor by flow cytometry using a modified method for preparing chromosome suspensions
Authors: Manogaran P. S. , Pande Gopal 
Published: August 2005

AbstractPreparation of good chromosome suspensions is a crucial step in obtaining good flow karyotypes from different types of cells. Although several methods are in use, not all are suitable for all cell types. Primary cells from murine tissues are specially difficult to flow karyotype. We have modified the existing protocols for preparing chromosome suspensions from the Zajdela ascitic hepatoma (ZAH) cells of rat and have constructed their flow karyograms. Our karyograms show a distinct marker chromosome in these cells, which could not be resolved using other protocols. We have also used this method for preparing chromosomes from the bone marrow cells of the rat and have compared their flow karyograms with the ZAH karyotype. We expect that our protocol will be useful for studying other types of tumor and normal cells. © 1993 Wiley‐Liss, INc.
DNA content measurements and an improved idiogram for the Indian muntjac
Authors: Levy Howard P. , Schultz Roger A. , Ordonez José V. , Cohen Maimon M. 
Published: August 2005

AbstractThe Indian muntjac, an asiatic deer, has the lowest diploid chromosome number among mammals (female 2N = 6; male 2N = 7). Using flow cytometric quantification of propidium iodide‐stained cells, we determined the DNA content of muntjac cells to be 94% that of human. This suggests that the muntjac may serve as a model for investigation of karyotypic evolution and rearrangement. In order to facilitate future comparative gene mapping studies, computer‐aided analysis of digitized metaphase chromosomes allowed development of a detailed Indian muntjac G‐banded idiogram incorporating both ISCN‐type nomenclature and quantitative estimates of the size of each band and position. © 1993 Wiley‐Liss, Inc.
Identification of pig chromosomes in pig‐mouse somatic cell hybrid bivariate flow karyotypes
Authors: Bouvet Alain , Konfortov Bernard A. , Miller Nigel G. A. , Brown David , Tucker Elizabeth M. 
Published: August 2005

AbstractTo identify pig chromosomes in pig‐mouse somatic cell hybrids, dual‐laser flow karyotypes and GTG‐banded metaphase spreads of pig, mouse, and 7 pig‐mouse hybrid cell lines were compared. Pig chromosomes no. 1, 2, 5, 6, 10, 11, 13, 14, 16, 18, X and Y were tentatively assigned to individual peaks in the pig flow karyotype on the basis of DNA content vs. relative chromosome length. In the 7 hybrid cell lines, 7 out of 8 peaks distinct from those of the mouse cell line could be correlated with the presence of pig chromosomes no. 5, 9, 10, 11 or 16, 14, 15, and 18, whereas 1 peak appeared to correspond to the presence of 1 middle‐size chromosome (3, 4, or 7). Other pig chromosomes present in the hybrids could not be detected with certainty due to superposition with mouse peaks and mouse chromosome rearrangements. © 1993 Wiley‐Liss, Inc.
Kinetic analysis of cytotoxic lymphocyte—target cell interaction as quantified by dual parameter flow cytometry
Authors: Waniewski Jacek , Palucka A. Karolina , Porwit Anna 
Published: August 2005

AbstractThe kinetics of conjugate formation between leukemic cell lines (K562 and Daudi) and lymphokine‐activated killer (LAK) cells was studied. A flow cytofluorometry method using double immunofluorescence staining was applied. During the first 15 min of incubation of LAK effectors with leukemic targets, a rapid binding occurred, followed by a plateau phase lasting until 30 min of observation. A considerable, yet not statistically significant, between‐donor variability was noticed. A mathematical model of conjugate formation kinetics, based on the analogy to enzyme kinetics, was formulated and validated. Parameters of the model were related to the binding capacity of effector and target cells, and to the lifetime of conjugates and free cells. The concordance of theoretical curves with experimental data proved that the described model can be considered as a useful tool for the evaluation of kinetic and dynamic characterization of conjugate formation between leukemic targets and LAK effectors. © 1993 Wiley‐Liss, Inc.
Quantitation of simian virus 40 T‐antigen correlated with the cell cycle of permissive and non‐permissive cells
Authors: Lehman John M. , Friedrich Thomas D. , Laffin Judith 
Published: August 2005

AbstractThese studies examined cell cycle progression and quantitative changes in T‐antigen following infection by SV40. Single cells were assayed by multiparameter flow cytometric analysis (FCM) for DNA content and T‐antigen expression. Conditions were used which permitted permissive, semi‐permissive, and non‐permissive cells to be monitored through two rounds of DNA synthesis induced by SV40. The permissive cells included the monkey kidney cell lines; CV‐1, Vero and BSC‐1 and the COS‐1 and COS‐7 which are CV‐1 cells transformed with an origin defective SV40. The non‐permissive cell strains included mouse embryo fibroblasts, Chinese hamster fibroblasts, and IMR‐90, a human diploid fibroblast. Cell types differed in the maximal amount of T‐antigen expressed per cell. Additionally, all cell types expressed a limited quantity of T‐antigen for each cell cycle phase and the quantity increased in each successive phase. The level in each phase was increased only two‐fold when 100 times more virus was used. Thus, for an infected population the quantity of T‐antigen was dependent on cell cycle distribution. High levels of T‐antigen were not required for permissive infection; however, permissive cells were distinguished from non‐permissive cells by the G2 levels. Permissive G2 cells had more than double the T‐antigen content expressed in G1, while nonpermissive G2 cells had less than a two‐fold increase over G1 levels. The appearance of cells with tetraploid DNA content and the failure to undergo mitosis correlated to the higher T‐antigen levels in the G2 of the permissive cells. Two other strains of SV40, 776, and VA45 exhibit similar values for T‐antigen expression and movement into tetraploid DNA content. This study establishes the levels of T‐antigen correlated to the cell cycle and cell type. © 1993 Wiley‐Liss, Inc.