bluuebunny/crossref_metadata_2025_split huggingface dataset:
RNA and DNA content of isolated cell nuclei measured by multiparameter flow cytometry
Authors: Piwnicka Magdalena , Darzynkiewicz Zbigniew , Melamed Myron R. 
Published: August 2005

AbstractNuclei were isolated from various cell types including Chinese hamster ovary (CHO) and L1210 leukemia cell lines, primary cultures of fibroblasts, nonstimulated and stimulated human lymphocytes and mouse liver cells, by using different isolation techniques. The isolated nuclei were subsequently stained with acridine orange (AO) and their fluorescence was measured by flow cytometry. Various procedures designed to stain DNAversusRNA differentially with AO were tested, and the staining of isolated nuclei was compared with that of whole cells. Control incubations with RNase and DNase were performed to estimate in whole cells and in nuclei the contribution of DNA and RNA to the fluorescence intensity at the respective wavelength bands of maximum emission for DNA (F530) and RNA (F>600).Depending on the cell type, 10–20% of total cell RNase‐sensitive F>600is localized in the nuclei. The RNase‐resistant portion of F>600of isolated nuclei represents the stainability of DNA. Suppression of cell proliferation in subconfluent cultures results in a decrease in both whole cell and in nuclear RNA content. Nonstimulated lymphocyte nuclei have considerably lower RNA content than nuclei from lymphocytes stimulated by pokeweed mitogen. Two subpopulations of nuclei having the same (2C) DNA content but differing in RNA content, are present in mouse liver; the cells entering S phase originate from the high RNA population.
Replication kinetics of Chinese hamster chromosomes as revealed by bivariate flow karyotyping
Authors: Cremer C. , Gray J. W. 
Published: August 2005

AbstractIn this paper we show that the replication kinetics of individual chromosomes can be estimated by bivariate flow cytometric analysis of chromosomes isolated from 5‐bromo‐2′‐deoxyuridine (BrdUrd) treated cells. To study the timing of replication of chromosomes, Chinese hamster M3–1 cells were grown for different times in BrdUrd containing medium. Colcemid was added during the last 90 minutes of each BrdUrd labeling period. Chromosomes were isolated from the Colcemid blocked mitotic cells and stained with Hoechst 33258 (HO) and chromomycin A3 (CA3). Bivariate HO‐CA3 flow karyotypes were measured in a dual beam flow cytometer. Labeling cells with BrdUrd for 2, 2 1/2, 3, 3 1/2, and 4 hours before chromosome isolation resulted in progressive quenching of the HO fluorescence of chromosomes Y and 10, 11, M2 while the HO fluorescence of all other chromosomes was either not quenched or was only slightly quenched. The CA3 fluorescence of all chromosomes was slightly enhanced by BrdUrd treatment. A quantitative evaluation of the quenching of HO fluorescence suggested that: a) greater than 50% of HO binding DNA of chromosomes 10, 11, M2 is synthesized in the last quarter of S phase; b) the Y chromosome synthesizes more than 50% of HO binding DNA in the last tenth of S phase making the Y chromosome the latest replicating M3‐1 chromosome. All other chromosomes have synthesized greater than 50% of their HO binding DNA before the last third of S phase.
Adaptation of the ektacytometer to automated continuous pO2 changes: Determination of erythrocyte deformability in sickling disorders
Authors: Bessis M. , Feo C. , Jones E. , Nossal M. 
Published: August 2005

AbstractThe ektacytometer is a visco‐diffractometric method to quantitate erythrocyte deformability. A continuous‐flow system has been added to the basic ektacytometer to record automatically cell deformation as ektacytometer index, (EI) as a continuous function of oxygen tension. Samples from normal donors (AA), donors heterozygous for the sickle cell gene (AS), and homozygotes (SS) were studied with this modified ektacytometer. As expected, the normal red cells showed no change in EI upon deoxygenation. At low pO2, the homozygotes were quite rigid, and their EI rose upon reoxygenation. Erythrocytes from AS subjects, when studied under conditions of physiologic pH and osmolarity, showed essentially normal deformability characteristics. When the osmolarity was raised to 310 and the pH lowered to 7.1, however, the AS cells also became rigid at low pO2. The system was also employed to study the effect of potassium cyanate, a known anti‐sickling agent. By increasing hemoglobin‐oxygen affinity, this agent shifted the pO2 versus EI curve to the left; thus, for any given pO2, the sickle cell population was more deformable in the presence of potassium cyanate than in its absence.
A new flow chamber and processing electronics for combined laser and mercury arc illumination in an impulscytophotometer flow cytometer
Authors: Severin E. , Ohnemus B. , Kiegler S. 
Published: August 2005

AbstractA flow cytometer of the impulscytophotometer (ICP)‐type has been developed for two‐parameter flow cytometry. It uses two different methods of excitation simultaneously: an argon laser beam focused onto the cell stream within a modified flow chamber, and the light of a high pressure mercury (Hg) lamp using Köhler illumination. The two fluorescence signals are collected sequentially by the same photomultiplier tube. The signals are processed on a cell‐by‐cell basis using new hardwired analog signal processing electronics enabling their accumulation and display as two‐parameter frequency distribution histograms.
The significance of DNA measurements for the early detection of bronchial cell atypia
Authors: Ono J. , Auer Gert 
Published: August 2005

AbstractInvestigations in an experimental model system and in clinical material indicate that squamous bronchial carcinoma is regularly preceded by the occurrence of bronchial cells with DNA values exceeding significantly the normal 4c region. The studies also show that cytomorphological methods do not allow the discrimination between milder forms of atypia and nonspecific cellular alterations. We describe a combined cytomorphological and cytochemical diagnostic procedure which facilitates the distinction between atypical squamous cells and nonspecific cellular changes in the bronchial tree.
Simultaneous three color and electronic cell volume analysis with a single uv excitation source
Authors: Haskill Stephen , Becker Susanne , Johnson Tim , Marro Dominic , Nelson Kay , Propst Roy H. 
Published: August 2005

AbstractWe have adapted an Ortho ICP‐22 flow cytometer (Ortho Instruments, Westwood MA) for the simultaneous measurement of three independent fluorochromes and cell volume. This has been accomplished by the addition of a third photomultiplier tube and the development of a new electronic cell volume (ECV) flow cell. Cells are first analyzed as they pass through the 100 U ECV aperture and are then excited approximately 15 μsec later by the 365 nm mercury arc beam reflected by a 400 nm dicroic mirror. Independent blue, green and red signals can be associated by a delay circuit to the ECV signal from the same cell. We have developed this system as an aid in the analysis of tumor cell and macrophage heterogeneity and differentiation. The choice of stain combinations to be used is extremely flexible and permits the anlysis of a wide range of enzyme activities in conjunction with DNA/RNA and phagocytic probes. Data presented indicates the value of this approach in identifying the presence of plasminogen activator‐like activity in both tumor and inflammatory cells within a malignant effusion as well as the quantitative expression of a number of markers of macrophage differentiation. Although the described techniques have been developed on a mercury are instrument, they can be used equally well with cell sorters.
Flow sorting in the study of cell‐cell interaction
Authors: Schaap G. H. , Verkerk A. , Vijg J. , Jongkind J. F. 
Published: August 2005

AbstractUndifferentiated mouse teratocarcinoma cells were cocultivated with differentiated mouse endoderm cells in order to study the possible induction of teratocarcinoma cell differentiation.A difference in DNA content between the two cell types was experimentally introduced to enable the reisolation of the teratocarcinoma cells after cocultivation. Pseudotetraploid (2s) endoderm cell lines were produced from pseudodiploid (1s) cells by treatment of these cells with cytochalasin B and flow sorting of tetraploid cells, using Hoechst 33342 as a viable DNA stain, with subsequent cloning of sorted single cells.In model experiments, where mixtures of 1s teratocarcinoma and 2s endoderm cells were stained with Hoechst 33342, the teratocarcinoma cells could be reisolated with a purity of about 97%.After a cocultivation period of 24 days viable teratocarcinoma cells could be isolated from the cocultivation mixture with a purity of 95%. Two dimensional analysis of the protein pattern of these cells indicated that cocultivation did not induce a differentiated (endoderm) pattern.Therefore according to this analysis the teratocarcinoma cells were not induced to differentiate during a 24 day cocultivation period. The method described offers excellent possibilities for studying cell‐cell interaction in vitro.
Isovolumetric sphering of erythrocytes for more accurate and precise cell volume measurement by flow cytometry
Authors: Kim Young Ran , Ornstein Leonard 
Published: August 2005

AbstractTypical mammalian erythrocytes are biconcave disks. Flow cytometric light‐scattering measurements of red cell volume show large, orientation‐dependent variations in signal size. To eliminate errors caused by shape factors, we sphere erythrocytes isovolumetrically on an Auto‐Analyzer manifold (Technicon Instruments Corp., Tarrytown, N. Y.) using a carefully controlled ratio of concentrations of a salt of long chain sulfated fatty alcohol (e.g.), sodium dodecyl sulfate (SDS) to albumin in neutrally buffered isotonic saline, producing a suspension with at least 2 1/2 hours of stability with respect to cell volume, cell count and retention of hemoglobin. A two‐step manifold modification of this procedure is preferred. It uses the endogeneous serum protein and a first SDS‐saline reagent without added albumin at a low dilution, and further dilution with a second SDS‐saline containing a very low concentration of glutaraldehyde. This bypasses a serious shelf life problem with the albumin‐SDS reagent.Microhematocrit and Coulter electronic impedance measurements were correlated with light‐scattering measurements on our own flow cytometer system of unsphered, sphered, and sphered and fixed samples. These reveal marked superiority over unsphered samples (e.g., reduced coefficients of variation of cell volume and improved correlation with microhematocrit) of the sphered and of the sphered and lightly fixed cells. The resulting tighter unimodal cell volume distributions also provide the following advantages: (1) The separation of signals of large platelets from that of the smallest red cells is substantially increased permitting better resolution of microcytic red cells in some abnormal bloods, and (2) bimodality of red cell volume distribution in abnormal bloods is more easily resolved.
A new method for cell volume measurement based on volume exclusion of a fluorescent dye
Authors: Gray Martha L. , Hoffman Robert A. , Hansen W. Peter 
Published: August 2005

AbstractSeveral methods currently in use for measuring mean corpuscular volume include: centrifuged packed cell volume, electronic impedance, and light scattering methods. Although these techniques are widely used and accepted, there are problems inherent to each method which may produce systematic errors that are difficult to estimate. This paper describes a new flow cytometric method of cell volume determination, based on the principle of volume exclusion, which may overcome the systematic errors of the methods currently in use. This method requires that the cells be suspended in a fluorescent dye which is unable to penetrate the cell membrane. The level of fluorescence which is produced when a narrow stream of the cell suspension is excited by a focused laser beam will remain constant until a cell arrives in the illuminated region thereby causing a decrease in fluorescence which is directly proportional to the cell's volume. The volume exclusion method is shown to give an estimate of mean red cell volume which correlates well with existing methods.
In‐use evaluation of the omnicon automated tumor colony counter
Authors: Herman Chester J. , Pelgrim Olga E. , Kirkels Wim J. , Verheijen Rene , Debruyne Frans M. J. , Kenemans Peter , Vooijs G. Peter 
Published: August 2005

AbstractThe reproducibility and accuracy of the Omnicon (Bausch and Lomb Inc., Rochester, NY) automated tumor colony counter for counting tumor colonies growing in double layer soft agar is evaluated and the reproducibility is compared with manual tumor colony counting. Replicate within day run‐to‐run colony counts of the Omnicon show a median correlation coefficient (r) of >0.985, and day‐to‐day median r of >0.980. In contrast, for manual colony counting, the best intra‐observer reproducibility achieved is a r of 0.943 and the best inter‐observer reproducibility is a r of 0.831. Analysis of results from individual culture plates counted by the Omnicon on 5 separate days shows a median coefficient of variation of 10% with 77% of the culture dishes showing coefficients of variation of colony counts over 5 days of less than 20%. Counting of culture plates during incubation shows that the Omnicon is counting tumor colonies developing after plating of a single cell suspension.
Cells on microspheres: A new technique for flow cytometric analysis of adherent cells
Authors: Bloch Donald B. , Smith Brian R. , Ault Kenneth A. 
Published: August 2005

AbstractTechnical problems have previously prevented the application of fluorescence activated cell sorting to the study of adherent cell populations. We have developed a procedure for attachment of human monocytemacrophages to 14–20 micrometer microspheres. These adherent cells on microspheres retained phagocytic capacity, could be stained for cell surface antigens using indirect immunofluorescence, and could be maintained in long‐term culture. They could be examined and sorted on a fluorescence activated cell sorter while still in the adherent state. This technique facilitates the flow cytometric study of adherent cells and permits the isolation of subpopulations of these cells.
Human T lymphocyte differentiation antigens: Effects of blood sample storage on LEU antibody binding
Authors: Hensleigh Paul A. , Waters Virginia Bryan , Herzenberg Leonard A. 
Published: August 2005

AbstractCurrent studies of human T lymphocytes and their subsets often use quantitative immunofluorescence analysis with monoclonal antibodies against cell surface antigens. With storage of whole blood or separated mononuclear cells for more than a few hours we have found marked changes in lymphocyte analysis using a fluorescence activated cell sorter (FACS). Experiments were done to determine if these lymphocyte changes were influenced by storage temperature and if lymphocytes could be made more stable by addition of culture media RPMI 1640 to whole blood. Optimal conditions found for blood storage were with with addition of 50% RPMI 1640 and with samples held at room temperature (22° C). With these storage conditions, delay on FACS analysis up to 24 hours did not result in spurious results. When blood samples are collected in places remote from the laboratory or when batch analysis of serially collected samples is desirable, excessive storage times should be avoided.
Direct determination of cell diameter, surface area, and volume with an electronic volume sensing flow cytometer
Authors: Schwartz A. , Sugg H. , Ritter T. W. , Fernandez‐Repollet E. 
Published: August 2005

AbstractA method is described in which the volume, surface area, and diameter of spherical cells can be quickly and easily determined from linear calibration curves using a flow cytometer which measures electronic volume and is equipped with a log amplifier.
Elastic and inelastic light scattering in flow cytometry
Authors: Kerker Milton 
Published: August 2005

AbstractA review of the fundamental aspects of elastic light scattering suggests that information about the shape and internal structure may best be obtained from signals measured in the backscattering directions. Size information can be most readily extracted from forward‐scattering signals. Spectral analysis of scattered signals with incident white light is a subject that merits further study.Signals from cells that have been stained with fluorescent dyes are proportional to dye content in the usual flow‐cytometric configuration except in the case of dense, strongly anisometric structures such as sperm cells.The recent discovery that Raman signals from molecules adsorbed on small silver particles are strongly enhanced suggests the possibility of utilizing this effect for identification of molecular species inside biological cells.
Morphometric synaptology of a whole neuron profile using a semiautomatic interactive computer system
Authors: Saito Kiichiro , Niki Kazuhisa 
Published: August 2005

AbstractWe propose a new method of dealing with morphometric synaptology that processes all synapses and boutons around the HRP marked neuron on a large composite electron micrograph, rather than a qualitative or a piecemeal quantitative study of a particular synapse and/or bouton that is not positioned on the surface of the neuron. This approach requires the development of both neuroanatomical procedures, by which a specific whole neuronal profile is identified, and valuable specialized tools, which support the collection and analysis of a great volume of morphometric data from composite electron micrographs, in order to reduce the burden of the morphologist.The present report is also concerned with the total and reliable semi‐automatic interactive computer system for gathering and analyzing morphometric data that has been under development in our laboratory. A morphologist performs the pattern recognition portion by using a large‐sized tablet digitizer and a menu‐sheet command, and the system registers the various morphometric values of many different neurons and performs statistical analysis.Some examples of morphometric measurements and analysis show the usefulness and efficiency of the proposed system and method.
Hoechst 33342 staining of mouse bone marrow: Effects on colony‐forming cells
Authors: Van Zant Gary , Fry C. Glenn 
Published: August 2005

AbstractWe have systematically studied the effect on hemopoietic colony‐forming cells of staining cellular DNA with the bisbenzimidazole dye, Hoechst 33342. Mouse bone marrow cells could be adequately stained in a 30‐60 min incubation with a 5 μM concentration of stain. Flow‐cytometric analysis of stained cells provided cell distributions with coefficients of variation for the G1 peaks of 6% or less under these conditions. We found considerable heterogeneity among hemopoietic colony‐forming cells with respect to the toxicity of the dye. Toxicity in the proliferatively quiescent stem cell population was not changed when the population became proliferatively active. In the sequence of most sensitive to least sensitive, the five progenitors studied could be arranged as follows: CFU‐M, a megakaryocyte colony‐forming cell; CFU‐E, a relatively differentiated erythroid precursor; BFU‐E, a primitive erythroid precursor; CFU‐GM, a granulocyte‐macrophage precursor; and CFU‐S, the spleen colony‐forming cell or hemopoietic stem cell. A staining procedure involving a 30‐min exposure to 5 μM Hoechst 33342 provided optimal staining and no loss in four of the five progenitor populations; the CFU‐M population was diminished by about 50%. We conclude that Hoechst can be regarded as a vital DNA stain for most bone marrow precursor populations, including the hemopoietic stem cell.
Ligand/receptor internalization: A kinetic, flow cytometric analysis of the internalization of N‐formyl peptides by human neutrophils
Authors: Finney David A. , Sklar Larry A. 
Published: August 2005

AbstractFluorescence flow cytometry was used to measure the internalization of the fluorescent ligand N‐formyl‐nle‐leu‐phe‐nle‐tyr‐lys‐fluorescein by human neutrophils. The internalization process was monitored by the accessibility of the receptor‐bound fluorescent ligand to quenching following a change in the pH of the extracellular medium from 7.4 to 3.0. In such a pH change, extracellular ligand or fluorescein are quenched immediately (excitation 488 nm). In contrast, intracellular fluorescein (derived from fluorescein diacetate) or intracellular ligand are quenched with half‐times of ∼20 or ∼40 sec, respectively, at 37°C. The fraction of internalized ligand is calculated by resolving the fast and slow components of the quenching process.Temporal resolution of the internalization process in this system depends upon two factors. We have previously shown that it is possible to examine essentially continuously the kinetics of ligand binding in the nM concentration range without removing the free ligand (Sklar LA, Finney DA, Cytometry 3:161, 1982). We have now modified a Becton Dickinson FACS IV sample head assembly to permit direct addition of reagents into the cell suspension while on‐line. This enables us to change the suspension pH and evaluate internalization with a time resolution of a few seconds. We observe that internalized ligand can be detected within 1 min and that the rate is proportional to the number of receptors occupied. The rate is essentially linear over the first few minutes and ∼60% of the receptor‐bound ligand is internalized after 3 min.
Flow‐cytometric monitoring of intracellular flavins simultaneously with NAD(P)H levels
Authors: Thorell Bo 
Published: August 2005

AbstractA cytofluorimeter is described, using the combination of Argon UV (351–363 nm) and Argon Blue (488 nm) lasers. The dual excitation makes it possible to monitor simultaneously the redox state of flavins and NAD(P)H as indicative of cell metabolic state. Light scatter, absorption, and staining with exogenous fluorescent dyes can add additional information. Thus, a five‐parameter flow analysis becomes possible. The present paper describes flavin and NAD(P)H measurements on isolated rat liver cells and mouse bone marrow.
Somatic cell genetics and flow cytometry
Authors: Kamarck Michael E. , Barbosa James A. , Kühn Lukas , Peters Pamela G. Messer , Shulman Lester , Ruddle Frank H. 
Published: August 2005

AbstractHuman genes coding cell surface molecules can be introduced into mouse host cells using a variety of somatic cell genetic techniques. Because these human gene products can be detected using indirect immunofluorescene on viable cells, the genes themsleves can be monitored and manipulated using flow cytometry and sorting. In this paper, we review ways that we have used cell sorting to develop a somatic cell genetic analysis of the human cell surface.