bluuebunny/crossref_metadata_2025_split huggingface dataset:
Flow cytometric electronic direct current volume and radiofrequency impedance measurements of single cells and particles
Authors: Hoffman R. A. , Johnson T. S. , Britt W. B. 
Published: August 2005

AbstractA flow‐system instrument is described that uses direct current and radiofrequency (rf) currents to detect simultaneously the low and high frequency impedance changes produced by biologic cells or particles suspended in saline traversing through a 93‐μm diameter sensing orifice. For nonmembranous particles and plastic microspheres both the Coulter direct current volume and rf‐parameter signals are proportional to particle volume. Cells having an intact plasma membrane produce rf‐impedance changes dependent additionally on the electrical properties of the plasma membrane and intracellular structures. Thus, biologically different cells having the same Coulter direct current volume can be distinguished if they differ in their electrical rf impedance properties.
An immunofluorescence method for monitoring DNA synthesis by flow cytometry
Authors: Gratzner H. G. , Leif R. C. 
Published: August 2005

AbstractAn immunofluorescence method for monitoring DNA synthesis in single cells has been developed for flow cytometry. With antiserum which is specific for 5‐bromodeoxyuridine (BrdUrd) and a second fluorescent label, BrdUrd‐incorporation pulses of 30 min are detectable. The fluorescence intensity of the incorporated BrdUrd, as determined by immunofluorescence, is related to the amount of BrdUrd incorporated, as shown by isotopic methods and cell sorting. Thus, the technique may be applicable to determining rates of replication per cell. Multiple samples of as few as 1 × 105 cells can be fixed, hydrolyzed and treated with the anti‐BrdUrd antiserum. Nuclear‐bound IgG is localized by fluorescein‐labeled avidin‐D. Since the technique uses whole cells, other parameters such as light scatter and DNA content can be simultaneously monitored so that cohorts of “labeled” cells can be followed through the cell cycle.
Clinical application of flow cytometry: A review
Authors: Laerum Ole Didrik , Farsund Tore 
Published: August 2005

AbstractA survey of the use of flow cytometry for clinical purposes is given. In the last decade the main clinical application of this technique has been measurements of cellular DNA content for estimation of cell cycle distribution and ploidy studies. A large body of data is now available on the presence of aneuploidy in different malignant diseases. By measurements with high resolution, the demonstration of abnormal cellular DNA content in several types of tumors can be of definite diagnostic value when combined with conventional diagnostic procedures. The prognostic significance of different types of DNA aberrations is so far not established. Attempts to monitor cancer treatment by studying altered cell cycle distribution have not been successful, although some applications are of potential value. The main reasons for this are the complexity of tumor tissue as well as difficulties with interpretation of altered cell cycle distribution caused by drug combinations. For further progress in this field more emphasis on other cell constituents than DNA measured by flow cytometry is desirable, either as single or as multiparameter measurements.
Dual laser beam focussing for flow cytometry through a single crossed cylindrical lens pair
Authors: Watson James V. 
Published: August 2005

AbstractTwo laser beams (argon 488 nm blue, and krypton UV lines) have been focussed either to the same point or to two different points in a vertical plane in a multiparameter flow cytometer. The technique exploits the focal length shortening of the longer wavelength beam that occurs with spherical and astigmatic lens aberrations. Ethidium bromidestained DNA is excited by both the argon 488 nm and krypton UV lines and the excitation intensities of both beams were set to give the same red fluorescence pulse height from ethidium bromide‐stained nuclei. DNA histograms were obtained for sequential focussing of the two beams and these were virtually identical irrespective of which beam was the first excitor and irrespective of which beam the histogram was recorded from. Coincident focussing of the two exciting beams resulted in a histogram of double the emission intensity.
Flow cytometric analysis of the uptake of hoechst 33342 dye by human lymphocytes
Authors: Szabó Gábor , Kiss Attila , Damjanovich Sándor 
Published: August 2005

AbstractHuman peripheral blood lymphocytes have been shown to resist staining with the DNA binding fluorochrome Hoechst 33342 by the cellular membrane. The rate of uptake of the dye is strongly temperature‐dependent with minimal uptake rate below 16°C. The activation energy of dye transport was found to be 135 kJ/mol above 20°C and about 20 kJ/mol below 16°C. Metabolic inhibitors accelerated, instead of inhibiting, the transport of the dye. Dead cells have been shown to stain promptly in contrast with the gradually staining viable cells. The uptake process in the vital staining conditions is suggested to involve a carrier mediated mechanism. Application of Hoechst 33342 as a fluorescent indicator of viability is proposed.
Flow fluorometric study of DNA content in nonproliferative Euglena gracilis cells and during proliferation
Authors: Bonaly J. , Mestre J. C. 
Published: August 2005

AbstractEthanol‐fixed Euglena gracilis cells have been analyzed by flow microfluorometry during the lag, logarithmic and stationary phases. The histogram of a plateau stage culture reveals, as expected, an unimodal distribution, but the peak is at a lower fluorescence intensity as compared to G1 logarithmic cells. The fluorescence intensity drops as the cells enter the stationary stage. Ultimately the decrease represents a change of about 25%. When cells recover from the plateau stage, the fluorescence intensity increases during the lag phase, and climbs to the level found in a Gl logarithmic population. The reason for the decrease in the fluorescence intensity during the stationary stage may be due to a possible loss of DNA or to a decrease in the number of chromatin‐binding sites for intercalating ethidium bromide.
Flow cytometric analysis of cellular endogenous fluorescence simultaneously with emission from exogenous fluorochromes, light scatter and absorption
Authors: Thorell Bo 
Published: August 2005

AbstractA cytofluorimeter capable of simultaneous 4‐parameter analysis (i.e., endogenous fluorescence (NAD(P)H), exogenous fluorescent dyes, absorption and small angle scatter) is described. Analyses have been performed on isolated rat liver cells, white blood cells, bone marrow, thymus cells and yeast under different metabolic states. This allows correlations between cell function and concentration of cell substances, size, refractive indices, etc. Kinetic measurements are also possible.
The application of flow cytometry for the quantification of the response of experimental tumors to irradiation
Authors: Beck H.‐P. , Brammer I. , Zywietz F. , Jung H. 
Published: August 2005

AbstractR1H rhabdomyosarcomas of the rat were irradiated with 15 Gy of x‐rays and the depopulation and repopulation of the tumor was determined by means of flow cytometry, quantitative histology and colony assays. The results show that flow cytometry yields important information about the cell population kinetics of irradiated tumors, but misleading results are obtained if flow cytometry is not combined with the determination of the numerical density of cells.
Expression of tumor‐associated antigens and kinetic profile of two in vitro human melanoma cell lines
Authors: Badaracco G. , Corsi A. , Maisto A. , Natali P. G. , Starace G. , Zupi G. 
Published: August 2005

AbstractThe growth characteristics and the antigenic profile of two human melanoma established cell lines in vitro are reported. The growth curves of the two cell lines show some peculiar features which are also detected in their DNA distribution content. In the late stages of growth, in fact, the cell DNA content resumes the initial distribution, suggesting a partial recovery of the cell proliferating potential. When the two cell lines were tested by indirect immunofluorescence with monoclonal antibodies to human melanoma associated antigens, a rather constant expression of these epithopes was observed.
Cell cycle kinetics of irradiated synchronous and asynchronous tumor cells with DNA distribution analysis and BrdUrd‐Hoechst 33258‐technique
Authors: Nüsse Michael 
Published: August 2005

AbstractRadiation‐induced progression delay in S‐phase and G2‐block, both depending on time of irradiation in the cell cycle, were measured in synchronized Ehrlich ascites tumor cells in vitro using flow cytometric analysis of DNA content in single cells. Similar results could be obtained by the BrdUrd‐Hoechst 33258 technique after irradiating asynchronous cells. BrdUrd replaces thymidine in newly synthesized DNA which is not stainable by the thymidine‐specific dye Hoechst 33258. The temporal development of the fluorescence distributions after addition of BrdUrd to the medium has been measured in the flow cytometer. In asynchronous cells the mean durations of the cell cycle phases and their perturbations after irradiation could be calculated, and the data agreed with the results in synchronized cells. Division delay after irradiation consists of a small progression delay in S‐phase and a block in G2‐phase. Both effects depend on time of irradiation during the cell cycle with delay in S‐phase increasing in irradiated late S‐cells and G2‐block increasing in irradiated G2‐cells. Data from both types of experiments were used with a simple model describing the fractions of cells in the cell cycle phases after irradiation had been applied.
The effect of abnormal cell proportion on specimen classifier performance
Authors: Castleman Kenneth R. , White Benjamin S. 
Published: August 2005

AbstractIn two previous papers we developed formulas relating the performance (error rates) of a two‐class specimen classifier to the performance of a preceding two‐class classifier and the number of cells examined (K. R. Castleman and B. S. White, Analytical and Quantitative Cytology 2:117, 1980 and K. R. Castleman and B. S. White, Cytometry 1:156, 1980.). This analysis assumed a certain proportion (p) of abnormal cells on an abnormal specimen. In this paper we examine what happens when a system designed assuming one value of p is presented with a positive specimen having a different abnormal cell proportion. We show that the specimen false negative error rate increases dramatically as p decreases below the design value, and conversely. This suggests that the specimen classification performance of a particular system should be quoted only with reference to the abnormal cell proportion of the specimens used for testing.
Proliferation kinetics of perturbed cell populations determined by the bromodeoxyuridine‐33258 technique: Radiotoxic effects of incorporated [3H]thymidine
Authors: Beck Hans‐Peter 
Published: August 2005

AbstractA simple analytical method is described for the evaluation of flow cytometric data of perturbed cell populations obtained by applying the BrdUrd‐33258 Hoechst technique. This procedure allows for determining the growth curve, the efflux from and the influx into G2 + M as well as the transit characteristics of cell cohorts through the G2 + M phase. As an example of the application of the method, the radiotoxic effects of incorporated 3H‐thymidine on the proliferation of L‐929 cells in vitro are described.
Enrichment of viable lymphocytes in defined cycle phases by sorting on the basis of pulse width of axial light extinction
Authors: Monroe John G. , Havran Wendy L. , Cambier John C. 
Published: August 2005

AbstractA correlation between cell size and cycle state is demonstrated for mitogen stimulated murine lymphocytes. Cell cycle analysis was performed using multiparameter cytofluorometric analysis of acridine orange stained cells. Cell size was determined by measuring “time of flight” of axial light extinction. Cells with diameters of 5.0–6.0 μm are shown to be predominately in G0. Among stimulated cells, those 6.0–7.0 μm in diameter are predominately early and late G1 (80%) with some S phase cells. Cells in G2 + M were found to predominate among lymphocytes with diameters of 7.5–12 μm. This approach provides a method for isolation of lymphocytes in specific phases of the cell cycle which are suitable for subsequent functional studies.
Flow cytometric analysis of RNA content in different cell populations using pyronin Y and methyl green
Authors: Pollack Alan , Prudhomme David L. , Greenstein David B. , Irvin George L. , Claflin Alice J. , Block Norman L. 
Published: August 2005

AbstractPyronin Y (PY) was used, in flow cytometric (FCM) systems, to estimate the RNA content per cell in formalin fixed EL4 leukosis tumor cells, enzyme dispersed R3327‐G rat prostatic adenocarcinoma cells, mouse spleen cells stimulated with concanavalin A, and human peripheral blood lymphocytes stimulated with phytohemagglutinin. Preincubation of the cells with methyl green (MG) blocked PY binding to DNA such that the intracellular fluorescence from MG‐PY was due primarily to its binding to RNA. Treatment of the cells with ribonuclease resulted in a 3‐ to 5‐fold reduction in the fluorescence intensity of intracellular MG‐PY.Mitogen stimulation of either mouse or human lymphocytes resulted in an increase in DNA (propidium iodide fluorescence) and RNA (MG‐PY fluorescence) content per cell over resting levels. Further, the changes in stimulated human lymphocyte DNA and RNA contents following 24, 48, and 72 hr of cell culture were monitored. The results showed that RNA levels were significantly increased prior to that of DNA. Also, the effects of different cell cycle phase specific blocking agents on lymphocyte cell cycle traverse were investigated. We found that: (a) actinomycin D inhibited the increases in cellular RNA and DNA; (b) hydroxyurea inhibited the increases in cellular DNA whereas increases in cellular RNA were only slightly reduced; (c) tritiated thymidine caused an accumulation of cells having high DNA and RNA contents; and (d) Colcemid promoted an accumulation of cells having high DNA contents while causing a reduction of cells having high RNA contents. These results were nearly identical to reports by other investigators using the metachromatic dye acridine orange to quantitate RNA per cell. Thus, the MG‐PY technique described is indicated to provide a stable and accurate measure of RNA content per cell.
Detection of transformed cells using a fluorescent probe: The molecular basis for the differential reaction of fluorescamine with normal and transformed cells
Authors: Parry Gordon , Blenis John , Hawkes Susan P. 
Published: August 2005

AbstractNormal and transformed fibroblasts can be discriminated by a flow cytometry assay on the basis of their differential reaction with fluorescamine. The cause of altered reactivity of transformed cells with this fluorescent probe has been investigated by a detailed analysis of its reaction with chicken embryo fibroblasts transformed by a temperature sensitive mutant of Rous sarcoma virus. The subcellular distribution of fluorescent adducts characterized by cell fractionation and gel electrophoresis procedures supports the hypothesis that transformed cells possess a surface barrier which decreases the accessibility of fluorescamine to reactive macromolecules. The barrier has been identified as being composed at least partly of hyaluronic acid, because of the ability of purified and specific hyaluronidase (from Streptomyces hyalurolyticus) to modulate the response of transformed cells to fluorescamine. Enzyme treatment of transformed cells prior to reaction with fluorescamine causes them to resemble nontransformed cells both in the nature of components labeled and in their fluorescence intensity.It is suggested that fluorescamine monitors an altered surface hyaluronic acid composition which occurs upon transformation. Its significance is discussed in terms of the known physical properties of the molecule and the finding that it is an early event in the process of transformation.
A follow‐up study of the progression or spontaneous regression of leukemia in mouse strains
Authors: Szöllösi J. , Ember I. , Trón L. , Kertai P. 
Published: August 2005

AbstractC3H/He‐mg and AKR mice were injected intraperitoneally with lymphocytes prepared from the spleen of (C3H/He‐mg X AKR)F1 hybrid mice bearing Gross‐virus leukemia. Lymphocyte suspensions were prepared from the spleen, mesenteric lymph node and thymus of mice at different days after the injection and analyzed for the ratio of leukemic to normal lymphocytes with a flow cytometer. The size and the fluorescein content of the cells incubated with fluorescein diacetate served for the distinction between normal and leukemic lymphocytes. After injection with leukemic lymphocytes, lethal leukemia developed in the AKR strain, whereas the C3H/He‐mg strain was characterized by a spontaneous regression after a transient development of leukemia. The percentage of leukemic lymphocytes was the highest in the spleen and it was the lowest in the thymus regardless of the stage of the disease. In the thymus of C3H/He‐mg mice the leukemic lymphocytes could be detected only during the crisis period.
Comparison of two flow cytometric assays for cellular RNA—acridine orange and propidium iodide
Authors: Wallen C. Anne , Higashikubo Ryuji , Dethlefsen Lyle A. 
Published: August 2005

AbstractTwo flow cytometric assays for cellular RNA, two‐step acridine orange (TSAO) and propidium iodide (PI), were compared with each other and with ultraviolet (uv) spectrophotometry of RNA to determine their ability to quantitate cellular RNA and to differentiate between proliferating and quiescent (Q) cells. The model system used for these comparisons was cells from unfed cultures of two mouse mammary tumor lines designated 66 and 67. The growth kinetics of unfed 67 and 66 cells were characterized by a decrease in cellular RNA (a factor of 2) with the decrease occurring throughout the entire population of cells as they entered plateau phase. The time course of the RNA decrease as monitored by the PI assay closely corresponded to that observed by uv spectrophotometric measurements. The TSAO assay, however, agreed with the uv spectroscopy and PI assays on the extent of the RNA decrease but showed no decrease in RNA until 24 hours after the other assays indicated that a significant decrease had occurred. The plateau cells from 67 and 66 unfed cultures had a greatly lowered RNA content and were predominately (>97%) in the G1 phase of the cell cycle in terms of DNA content. However, when compared with exponentially growing cells, they were a distinct Q population. This distinction could be observed using either the TSAO or the PI assay. The TSAO assay provided better resolution of the Q population and has the added advantage of giving DNA distribution simultaneously. Therefore, both flow cytometric assays are particularly useful in this system; the TSAO for monitoring the Q population and the PI for determination of kinetic changes in total cellular RNA.
Analysis of glycosaminoglycans of flow sorted cells: Incorporation of [35S]sulfate and [3H]glucosamine into glycosaminoglycans of B16‐F10 cells during the cell cycle
Authors: Blair Owen C. , Burger Douglas E. , Sartorelli Alan C. 
Published: August 2005

AbstractThe incorporation of [35S]sulfate and [3H] glucosamine into cetyl pyridinium chloride (CPC) precipitable glycosaminoglycans was determined in B16‐F10 cultured cells sorted with respect to DNA content. Incorporation into surface material was measured indirectly as the difference between the radio‐activity of control and trypsin treated cells. Approximately 80% of the total cellular [35S] sulfate labeled CPC precipitable material, but only 5% of that labeled by [3H]glucosamine, was removed by this mild trypsin treatment. Incorporation of [35S]sulfate into the trypsin removable surface material increased progressively from G1 to S to G2 + M in both long‐term (48 hours) and shortterm (1 hour) labeled cells, while the ratio of surface to total incorporated [35S]sulfate remained relatively constant. Incorporation of [35S]sulfate into total cellular glycosaminoglycans in long‐ and short‐term labeled cells increased as cells progressed from G1 to S to G2 + M; the incorporation of [3H]glucosamine into CPC precipitable material also increased progressively from G1 to S to G2 + M in long‐term labeled cells but was greater during S phase relative to G1 or G2 + M in short‐term labeled cells. The degree of sulfation of glycosaminoglycans as represented by the ratio of [35S]sulfate to [3H]glucosamine of double labeled cells was relatively constant in long‐term labeled cells but was increased during the G1 and G2 + M phases of short‐term labeled cells. Comparison of the degree of sulfation of short‐term with long‐term labeled cells suggests that highly sulfated glycosaminoglycans may be turned over more rapidly during G1 and G2 + M phases of the cell cycle.
Flow sorting of mouse pancreatic B cells by forward and orthogonal light scattering
Authors: Nielsen O. , Larsen J. K. , Christensen I. J. , Lernmark Å. 
Published: August 2005

AbstractMouse islet cell suspensions were separated into subpopulations based on forward low angle and orthogonal scattered light. Separated fractions were analyzed for hormone content by radioimmunoassays. The pancreatic B‐cells had the highest orthogonal light scattering, while they overlapped in forward low angle light scattering with other endocrine (glucagon, pancreatic polypeptide and somatostatin containing cells) and nonendocrine cells. Living and paraformaldehyde fixed islet cell suspensions showed similar distributions of combined light scattering.
Determination of linear fluorescence intensities from flow cytometric data accumulated with logarithmic amplifiers
Authors: Muirhead K. A. , Schmitt T. C. , Muirhead A. R. 
Published: August 2005

AbstractLogarithmic amplifiers are useful in accumulating flow cytometric data with a large dynamic range. However, quantitative comparison of fluorescence intensities for different samples or different subpopulations within a sample is simplified by the conversion of data from log space back to linear space. A method is described in which fluorescent polystyrene spheres of differing intensities are used to construct a calibration curve for the logarithmic intensity scale. This allows calculation of relative linear intensity for each channel of the logarithmically accumulated data and determination of linear fluorescence means and coefficients of variation for comparative purposes. Fluorescent spheres of appropriate intensity may also be used as internal standards to monitor instrument and/or stain stability for samples accumulated using logarithmic amplifiers.