bluuebunny/crossref_metadata_2025_split huggingface dataset:
Physical discrimination between human T‐lymphocyte subpopulations by means of light scattering, revealing two populations of T8‐positive cells
Authors: Terstappen L. W. M. M. , De Grooth B. G. , Nolten G. M. J. , Ten Napel C. H. H. , Van Berkel W. , Greve J. 
Published: August 2005

AbstractLight‐scattering properties of human T‐lymphocyte subpopulations selected by immunofluorescence were studied. Based on differences in orthogonal light scattering, two subpopulations of T8‐positive cells can be distinguished. The first population (T8a) has the same orthogonal light‐scattering properties as T4‐positive cells, whereas the orthogonal light scattering of the second population (T8b) was about 70% larger. Orthogonal light scattering of Leu7‐positive lymphocytes resembles that of the T8b population. We have studied the occurrence of the subpopulation in healthy individuals and we discuss their possible functional identification. Light‐scattering properties of lymphocyte subpopulations in two patients with B‐cell chronic lymphatic leukemia suggest that this observation is of clinical interest.
Malignancy index based on flow cytometry and histology for renal cell carcinomas and its correlation to prognosis
Authors: Baisch H. , Otto U. , Klöppel G. 
Published: August 2005

AbstractFifty‐five human renal cell carcinomas, removed by nephrectomy, were classified using flow cytometry and histology. The parameters obtained by flow cytometry were DNA index, fractions of cells in the phases, and fractions of tumor cells versus normal cells. From histology, the routine classification of tumor structure and morphology were obtained, and a nuclear grading according to Arner et al. (Acta Chir Scand [Suppl] 346:1–12, 1965) was determined. All parameters including tumor stage according to Robson et al. (J Urol 101:297–301, 1969) were subjected to discriminant analysis in order to define a malignancy index. The patients were divided into two groups with good and poor prognosis (low and high risk). Those patients who were free of multiple metastases in 2 years and more after nephrectomy were assigned to the group “good prognosis”; those who developed multiple metastases for 2 years represented the “poor prognosis” group. The malignancy index determined by discriminant analysis yielded 91% correct predictions of prognosis.
A statistical analysis of prescreening alarms in a population of normal and abnormal gynecologic specimens
Authors: Wheeless L. L. , Robinson R. D. , Cox C. , Berkan T. K. , Reeder J. E. 
Published: August 2005

AbstractA multidimensional slit‐scan flow system has been developed to serve as an automated prescreening instrument for gynecological cytology. Specimens are classified abnormal based on the number of cells having elevated nuclear fluorescence (alarms). An alarm region in a bivariate histogram of nuclear fluorescence versus nuclear‐to‐cell‐diameter ratio is defined. Alarm region probability arrays are calculated to estimate the probability that an alarm falling in a particular bin of the alarm region is either from a normal or an abnormal specimen. From these arrays, a weighted alarm index is generated. In addition, summary indices are derived that measure how the distribution of alarms in each specimen compares with the average distributions for the normal and abnormal specimen populations. These indices together with current features are evaluated with respect to their utility in specimen classification using a nonparametric classification technique known as recursive partitioning. Resulting classification trees are presented that suggest information in the distribution of alarms in the bivariate histogram. In addition, they validate the features and rules currently used for specimen classification. Recursive partitioning appears to be useful for multivariate classification and is seen as a promising technique for other applications.
Flow cytometric analysis of hairy cell leukemia using right‐angle light scatter
Authors: Van Bockstaele Dirk R. , Berneman Zwi N. , Peetermans Marc E. 
Published: August 2005

AbstractWe report on an easy and reliable method for the enumeration of the typical cells present in the blood, bone marrow, and spleen of patients with hairy cell leukemia. Samples of five patients were analyzed. In three patients, the hairy cells were very accurately followed during treatment with alpha‐interferon, using right‐angle light scatter to differentiate them from other leukocytes present in total blood. Excellent correlation was obtained with the microscopic hairy cell enumeration. Additional verification by cell sorting and immunofluorescence studies confirmed these findings.
Binding of Hoechst 33258 to chromatin in situ
Authors: Stokke Trond , Steen Harald B. 
Published: August 2005

AbstractThe binding of Hoechst 33258 to rat thymocytes, human lymphocytes, and NHIK 3025 tissue culture cells was studied by measuring the fluorescence and light scattering of the cells as functions of dye concentration using flow cytometry. The results indicated that there were two different modes of binding of Hoechst 33258 to chromatin in situ at physiological pH. Type 1 binding, which dominated at total dye/phosphate ratios below 0.1 (0.15, M), was characterized by a binding constant of the order 107 M−1 and fluorescence with high quantum yield. Further binding of the dye resulted in a reduced blue/green fluorescence ratio, indicating that secondary sites were occupied. Binding at secondary sites above a certain density (0.1 ≤ bound dye/phosphate ≤0.2) induced strong quenching of fluorescence and precipitation of chromatin. Precipitation was quantitated by measuring the large‐angle (≥ 15°) light scattering of the cells above 400 nm, i.e., outside the Hoechst 33258/DNA absorption spectrum, as a function of dye concentration. In contrast, the light scattering at 365 nm, i.e., within the absorption spectrum of Hoechst 33258/DNA, was independent of the total dye/phosphate ratio. The coefficient of variation of the light‐scattering (≥ 400 nm) histograms decreased with Hoechst 33258 concentration. Type 2 binding to histone‐depleted chromatin was cooperative (Hill‐coefficient ≈ 2) and the apparent binding constant was 2–3 · 105 M−1 as determined from quenching and precipitation data. The final stoichiometry in the dye/chromatin complex calculated from quenching data was q = 0.70 (bound dye/phosphate ratio), while light‐scattering data yielded q = 1.35. The dye‐induced precipitation of intact chromatin of human lymphocytes was not cooperative (Hill‐coefficient ≈ 1.0). The precipitation of DNase 1 digested chromatin, however, was described by two‐component kinetics. The components had Hill‐coefficients of 1.0 and 2.4, respectively; the appearance of the latter was interpreted as reflecting precipitation of relaxed chromatin. Histone‐depletion increased the number of Hoechst 33258 type 1 binding sites in chromatin by about 30%, while the dye‐induced increase in light scattering decreased by about 40%.
Characterization of f‐Met‐Leu‐Phe‐stimulated fluid pinocytosis in human polymorphonuclear leukocytes by flow cytometry
Authors: Davis Bruce H. , McCabe Elizabeth , Langweiler Marc 
Published: August 2005

AbstractN‐formylated chemotactic peptide stimulation of human neutrophils initiates a number of cellular processes, such as lysosomal enzyme release and superoxide anion production, that are indicative of the events of neutrophil activation during the acute inflammatory response in disease. This study characterizes a newly recognized neutrophil activation event, N‐formylated chemotactic peptide‐stimulated fluid pinocytosis in human neutrophils, using a novel flow cytometric assay for this activity. Fluid pinocytosis was found to be inhibited by acidic pH and low temperature but could be enhanced by cytochalasin B treatment or surface adherence by neutrophils. The activity measured by this new assay of fluid pinocytosis appears to be separate and distinct from lysosomal enzyme release and receptor‐mediated adsorptive endocytosis in neutrophils. The physiologic significance of N‐formylated chemotactic peptide‐stimulated fluid pinocytosis is not known, but a possible relationship to neutrophil locomotion is discussed.
Measurement of myeloid maturation by flow cytochemistry in HL‐60 leukemia: Esterase is inducible, myeloperoxidase is not
Authors: Ross Dennis W. 
Published: August 2005

AbstractThe phenomenon of leukemic cell maturation requires a measurment of myeloid maturation to understand the process and to exploit it as a means of therapy for leukemia. The HL‐60 leukemic cell line was used as a model of induced leukemic cell maturation in order to develop a method of quantitating granulocytic and monocytic maturation in response to drug therapy. An automated flow cytochemistry system (Hemalog‐D) was employed to measure mean cell volume, myelo‐peroxidase (MPO), and nonspecific esterase (NSE). For granulocytic maturation induced by vitamin A or DMSO, MPO and cell volume decreased by 50%, maintaining a constant mean cellular MPO concentration throughout maturation from promyelocyte to neutro‐phil‐like forms. For monocytic maturation induced by low‐dose ARA‐c, the mean NSE increased substantially, while cell volume remained constant. Unlike MPO concentration, NSE was truly inducible and thus a useful quantitative measure of maturation caused by low‐dose ARA‐c. Flow cytochemistry and cytofluorometry may be developed to allow for quantitative monitoring of therapeutic trials of induced maturation in human leuke‐mias. However, this will require adapting these techniques to the complexity of human leukemias in vivo, and the necessity of handling heterogeneous populations encountered in bone marrow samples.
Modification of a laser‐based flow cytometer for high‐resolution DNA analysis of mammalian spermatozoa
Authors: Johnson L. A. , Pinkel D. 
Published: August 2005

AbstractModification of a Coulter EPICS V orthogonal laser‐based flow cytometer/cell sorter allows resolution of X and Y mammalian sperm populations based on DNA content. The modification consists of beveling the sample injection tube situated in the flow chamber, adding a second fluorescence detector directly forward along the laser beam axis, and routing the collected fluorescence through an optical fiber bundle to one of the existing photomultiplier tubes. The X and Y chromosome‐bearing spermatozoa from the bull, boar, and ram can be resolved using this system.
G2 arrest, binucleation, and single‐parameter DNA flow cytometric analysis
Authors: Ronot Xavier , Hecquet Christiane , Larno Suzanne , Hainque Bernard , Adolphe Monique 
Published: August 2005

AbstractOne important facet of flow cytometry involves the effects of pharmacological agents on cell cycle progression. Comparative G2 fraction perturbations were examined: effects of sodium butyrate on articular chondrocytes, effects of an antineoplastic agent (SOAz) and an antirheumatic drug (D‐penicillamine) on HeLa cells. Even though DNA flow cytometric analysis detects preferentially an induction of G2 arrest, the mode of action of these agents on the cell cycle is different. Sodium butyrate and D‐penicillamine lead to an increase of binucleate cells due to cytokinesis perturbation. Because of similar fluorescence intensity, distinguishing G2 from binucleate GO/1 cells is not easily possible using DNA content measurement and reflects a failure of flow cytometry in the detection of binucleate cells. Rapid cell cycle analysis of single cells should contribute greatly to the study of pharmacological interactions, but DNA flow cytometric measurements obtained from cultured cells exposed to certain agents must be cautiously interpreted because those may interact on cytokinesis and induce artefacts in histogram interpretation.
Accuracy and reliability of flow cytometric DNA analysis using a simple, one‐step ethidium bromide staining protocol
Authors: Petersen S. E. 
Published: August 2005

AbstractSources of variation and error were investigated for a simple flow cytometric analysis of DNA content of detergent‐isoiated nuclei stained with ethidium bromide. Using the ploidy classes of mouse liver nuclei, deviations from linearity were assessed for three different instruments. In more extreme settings, the maximum deviations for a FACS instrument were up to 6 to 9%, but in general deviations were around 1% or lower for all instruments. As biological DNA standards, human peripheral lymphocytes and trout erythrocytes appeared to be suitable and easy to store frozen. The erythrocytes had dyebinding characteristics similar to those of human lymphocytes and a 20% lower fluorescence, thus being well suited as an internal standard, as was demonstrated in tumor ploidy analyses performed with varied tissue concentration. Staining homogeneity was improved when staining time was extended to 24 h, at which time male and female lymphocytes were completely separated with an average difference in DNA content of 1.9%. A small difference in fluorescence between mitogen‐stimulated and unstimulated lymphocytes was reduced to less than 1% after 24 h of staining. In general, the manipulations of the conditions for the analysis resulted in maximum variations of around 1%, indicating the robustness and reliability of the technique.
The use of an image analyser in human tumour clonogenic assays
Authors: Kahn Edmond , Benard Jean , Di Paola Robert 
Published: August 2005

AbstractThe counting of tumoral colonies growing in semisolid media must be achieved in human tumour clonogenic assays. An image analyser can be advantageously used to overcome the limit of the eye when performing this task. Colonies growing from human cancer cell lines were analysed. Quantitative results on the cytotoxic effect of chemotherapeutic agents were obtained that could not be reached by means of the eye.
DNA and nuclear protein measurement in isolated nuclei of human endometrium
Authors: Oud Peter S. , Reubsaet‐Veldhuizen José A. M. , Henderik Jos B. J. , Pahlplatz Martin M. M. , Hermkens Huub G. , Tas Johan , James Jan , Peter Vooijs G. 
Published: August 2005

AbstractFeulgen‐DNA and nuclear light green‐protein measurements have been performed in isolated nuclei of normal (nonmalignant) and malignant human endometrial homogenates.The DNA content of the GO/G1 fraction of malignant endometrium showed much overlap with that of normal endometrium, or was slightly increased. Two of the 18 carcinomas were clearly aneuploid. No correlation was found between the histological grade and the DNA content. The tumors of clinical stage II and higher all had a higher DNA content than that of normal endometrium.The percentage of cells present in the proliferative fraction was higher in proliferative endometrium than in secretory and postmenopausal atrophic endometrium. For malignant endometrium, percentages were found comparable to that of normal endometrium or higher. No correlation was found with the histological grade. Tumors of stage II and higher had intermediate values compared to those of carcinomas below stage II.The nuclear protein/DNA ratio of malignant endometrium completely overlapped that of normal endometrium. However, for postmenopausal women, most values of the carcinomas exceeded that of normal, atrophic, endometrium. Within the tumor population, no correlation was found with the histological grade. Higher values were found with tumors of clinical stage II and higher.
DNA and nuclear protein measurement in columnar epithelial cells of human endometrium
Authors: Oud Peter S. , Reubsaet‐Veldhuizen José A. M. , Beck Hans L. M. , Pahlplatz Martin M. M. , Hesselmans Gerard H. F. M. , Hermkens Huub G. , Tas Johan , James Jan , Peter Vooijs G. 
Published: August 2005

AbstractPropidium iodide DNA flow cytometry, Feulgen‐DNA, and nuclear light green protein scanning cytometry were performed in columnar epithelial cells of normal, nonmalignant human endometrium and endometrial adenocarcinomas. Columnar cells were identified by immunohistochemical staining for cytokeratin 18, an intermediate filament protein specifically present in columnar cell epithelium. DNA measurements derived from flow and scanning cytometry showed comparable results. The DNA content of the G0/G1 fraction of the adenocarcinomas had a considerable overlap with that of normal endometrium, with that of the carcinomas shifted toward higher values. For the carcinomas, no correlation was found with the histological grade, with the exception of the adenosquamous carcinomas. Most of the clinical stage I tumors showed a DNA content in the normal diploid region. Three of the four carcinomas of clinical stage II and higher had an increased DNA content.For the carcinomas, the percentage of cells in the proliferative fraction, as determined from scanning cytometric derived DNA histograms, was comparable to that of normal endometrium, or higher. No correlation was found with the histological grade. Tumors of clinical stage II and higher had intermediate values compared to carcinomas of lower stages.The nuclear protein/DNA ratio of malignant endometrium completely overlapped that of normal endometrium. Within the tumor population, no correlation was found with the histological grade, with the exception of the adenosquamous carcinomas, and clinical stage.Based on the aforementioned parameters, no discrimination could be obtained between normal and malignant endometrium. However, when the DNA content of the G0/G1 fraction was combined with the coefficient of variation of the nuclear protein/DNA ratio, a clear discrimination could be obtained with only two false‐positive cases.
Flow cytometric analysis of liposome‐nuclei interaction: Transfer and intranuclear release of carboxyfluorescein
Authors: Papa Stefano , Caramelli Elisabetta , Allegri Giorgio , Billi Anna M. , Manzoli Francesco A. , Capitani Silvano 
Published: August 2005

AbstractFlow cytometric analysis of the transfer of liposome‐encapsulated carboxyfluorescein to isolated rat liver nuclei indicated that the fluorophore is actively taken up in this form, while negligible amounts are transferred when the dye is free in the reaction medium. The kinetic analysis of the uptake indicated a time‐ and dose‐dependent reduction of the slope in the absence of transport saturable sites on the nuclear surface and of quenching phenomena. The comparison between entire and membrane‐deprived nuclei demonstrated that the initial rate of uptake was higher in the absence of the complete nuclear envelope. The intranuclear binding sites were considered on the basis of the fluorescence distribution and of quantitative estimates of the amount of linked dye. The possibility of employing flow cytometry to monitor the interactions between liposomes and isolated nuclei by means of a fluorescent probe is discussed.
Flow cytometric estimation of transmembrane potential of macrophages—A comparison with microelectrode measurements
Authors: Jenssen Hans‐Ludwig , Redmann K. , Mix E. 
Published: August 2005

AbstractPotential‐dependent accumulation of the lipophilic cationic dye 3,3′ dihexyloxacarbocyanine (DiOC6(3)) in macrophages has been investigated. Resulting fluorescence of cells was measured by flow cytometry. Alterations of membrane potential of macrophages were induced by ionophore treatment (valinomycin and gramicidin) in a dose‐dependent (10−5 M‐107 M) and time‐dependent (0 min‐45 min) manner. Resulting changes in relative fluorescence intensity were compared with changes of transmembrane potential measured by intracellular recordings obtained by applying glass microelectrodes. The comparative studies offer the possibility to calibrate the flow cytometric estimate of membrane potential of suspended cells. Equilibration of dye partition between cells and surrounding medium is strictly potential‐dependent at dye concentrations between 5 × 10−8 M and 10−7 M and within an incubation interval from 10 min up to 30 min after addition of dye. Conclusions are drawn concerning the field of application of the optical method. Dynamics of electrical processes following ionophore treatment are discussed in terms of molecular mechanisms of altered ionic transport.
Flow cytometric measurement of cytoplasmic pH: A critical evaluation of available fluorochromes
Authors: Musgrove Elizabeth , Rugg Catherine , Hedley David 
Published: August 2005

AbstractThree pH‐sensitive fluorochromes–4‐methyl‐umbelliferone(4MU),2,3‐dicyano‐hydroquinone (DCH), and 2′,7′‐bis(carboxyethyl)‐5,6‐carboxy fluorescein (BCECF)–were evaluated for their resolution, range, and stability of cellular fluorescence. Flow cytometric techniques for determining cytoplasmic pH (pHi) have been fully described for 4MU and DCH; BCECF has previously been used for fluorimetric estimation of pHi, and was adapted to flow cytometry. For each fluorochrome, the ratio of fluorescence intensity at two wavelengths gives a measure of pHi, which may be calibrated by obtaining the fluorescence ratios for cells suspended in buffers of varying pH in the presence of a proton ionophore.Reliable calibration proved difficult using 4MU, partly because of poor retention within cells. Both DCH and BCECF could be calibrated using a fluorescence ratio and had resolutions of 0.2 and 0.4 pH units, respectively. The fluorescence of DCH is so strongly pH dependent that there were practical difficulties in its use over a wide pH range; however, pHi measurements are possible between pH 6.0 and pH 7.5 using either DCH or BCECF. Substantial dye leakage was found for 4MU and, to a lesser extent, DCH, while BCECF was retained by cells for up to 2 hours. Despite its lower resolution BCECF had a usable range of more than 1.5 pH units and this coupled with its stable fluorescence and excitation at 488 nm rather than UV suggests a wide application.
Analysis of intracellular antigens by flow cytometry
Authors: Jacobberger James W. , Fogleman David , Lehman John M. 
Published: August 2005

AbstractQuantitative immunofluorescence is routinely used in flow cytometric assay of cell surface antigens. Intracellular antigens have not been as tractable. Recent publications (Proc Natl Acad Sci 80:5573–5577, 1983; Cytometry 6:208–214, 1985) and the results presented here demonstrate that highly specific staining and subsequent quantitative analysis are not only possible but rather easily achieved. High purity antibodies and optimized fixing and staining technique are required. Under conditions presented in this paper, 97% of fluorescein specific signal is specific to the T antigen of SV40 when monoclonal antibody to this antigen is used with a transformed cell line. Three levels of quantitative analysis are discussed: 1) estimation of the fraction of positive cells in a mixed +/− population, 2) estimation of the average content of antigen in a population of cells, and 3) measurement of the distribution of antigen content within a population of cells. Results are presented that demonstrate that relatively low specific signal (measured as percentage of total signal) can be tolerated to achieve the first level and that the current methods available that produce a high specific signal are sufficient to achieve the second level. The third level will require further research aimed at lowering the variation introduced by the method of measurement.