bluuebunny/crossref_metadata_2025_split huggingface dataset:
Fixation of mammalian cells for flow cytometric evaluation of DNA content and nuclear immunofluorescence
Authors: Schimenti Kerry J. , Jacobberger James W. 
Published: August 2005

AbstractMammalian tissue culture cells were fixed with 3 different alcoholic fixatives—acetone:methanol, EtOH, and MeOH. The quality of the resulting DNA histograms was evaluated by comparison of CV, G1/G2 ratio, G1 mode, cell aggregation, and debris formation; 81–90% MeOH (final concentration) was determined to be the optimal fixative by these criteria. A procedure was then examined using a prefix with paraformaldehyde followed by MeOH (PF/MeOH). This procedure produced cell preparations with reduced debris and aggregation, equivalent mode and ratio, but increased CV when compared with MeOH fixation. Both MeOH and PF/MeOH fixation procedures were then compared for their utility in dual staining for DNA and intracellular immunofluorescence for a nuclear protein, SV40 T antigen (Tag). Since alcohols are known to affect immunofluorescence staining of some antigens, fixation with paraformaldehyde followed by Triton X100 permeabilization (PF/TX) was also included in this comparison to generalize the study by providing an alternative to MeOH permeabilization. The three procedures were evaluated for the quality of the sample by measuring the same descriptors of the DNA parameter as in the alcohol study. PF/TX consistently produced samples with decreased DNA CV and less debris and aggregation compared to MeOH methods. Two criteria were used to evaluate immunofluorescence–the amount of Tag measured and reproducibility. All MeOH methods were equivalently reproducible with CV's less than 3%. PF/TX was slightly less so with a CV of less than 6%. In contrast, different levels of Tag were measured for each procedure. For mouse 3T3 cells infected with a recombinant retroviral vector encoding T antigen, the level of T antigen measured after PF/MeOH was 21% greater than in MeOH fixed cells, and the level in PF/TX fixed cells was 37% less. The fraction of fluorescence specific to T antigen for these cells was 79–83% for all procedures. The lower levels measured after fixation by PF/TX were shown to be due to epitope masking. Why higher levels are measured with PF/MeOH procedures is unknown at present but may be due to antigen retention. Therefore, each of these fixation methods may be used with confidence in reliability but they are not equivalent with respect to the molecular architecture of the nucleus. It is postulated that PF/TX permeabilizes cells but cells retain native supramolecular structure, whereas MeOH based fixatives disrupt this structure and randomize availability of epitope to antibody. If so, the two procedures could be used as complementary procedures to study gene expression and function.
Increase in acridine orange (AO) fluorescence intensity of monocytes cultured in plastic tissue culture plates as measured by flow cytometry
Authors: Myc Andrzej , Pizzolo John G. , Dygulski Krzysztof , Melamed Myron R. 
Published: August 2005

AbstractAlthough the green‐red fluorescence of AO is an accepted measure of DNA‐RNA content, respectively, it is actually a measure of the fluorescence of dye bound to nucleic acids, and may vary with changes in accessibility to the dye. It has been shown for example that extraction of nuclear proteins results in a marked increase in DNA stainability. Moreover, in certain cell systems the binding of fluorochromes correlates with structural modifications in chromatin that accompany cell differentiation. We report here that changes in green & red fluorescence intensity also occur in long‐term monocyte cultures. The increased red fluorescence intensity observed in cultured monocytes may reflect ribosomal RNA synthesis and the increased green fluorescence enhanced AO accessibility to DNA due to changes in chromatin organization. We compared cultured monocytes from bladder cancer patients and healthy donors. The results indicate a small but statistically significantly greater increase in mean green & red fluorescence of cultured monocytes from the cancer patients. These fluorescence variations may indicate differences in the immunologic status of cancer patients and/or be related to disease state.
HOME: Highly optimized microscope environment
Authors: Brugal Gérard , Dye Richard , Krief Bruno , Chassery Jean‐Marc , Tanke Hans , Tucker James H. 
Published: August 2005

AbstractThe Highly Optimized Microscope Environment (HOME) is a computerized microscope designed to assist pathologists and cytotechnicians in clinical routine tasks. The prototype system consists of a IBM‐PC compatible computer and a light microscope in which a built‐in highresolution computer display image is superimposed on the optical image of the specimen. Also, a manually operated encoding stage and objective turret encoder are used to provide continuous monitoring of the stage coordinates and microscope magnification to the computer. This allows any position on a slide to be uniquely defined and makes it possible to measure interactively lengths and areas larger than the size of the microscope field. Software, written in the C language and operating under the MS‐DOS/MS‐Windows environment, is controlled by means of a mouse‐driven cursor moving over menu light‐buttons displayed on the microscope image. The HOME microscope workstation is potentially useful in a wide range of applications such as (i) tagging information on particular cells and tissue structures that can thus be accurately located and relocated, (ii) performing morphometric measurement, differential counting, and stereological assessment of biological specimens, and (iii) training and educating laboratory personnel. Finally, HOME will offer in the near future a userfriendly interface for automatic image processing of cells and tissue entities in interactively selected specimen areas.
Expression of proliferation associated antigens in the cell cycle of synchronized mammalian cells
Authors: Bolton Wade E. , Mikulka Warren R. , Healy Cynthia G. , Schmittling Robert J. , Kenyon Norma S. 
Published: August 2005

AbstractFlow cytometric bivariate analysis was used to investigate the expression of PCNA, p120 and p145 during the cell cycle of a mammalian cell line (CHO‐K1). Initially, aliquots of cells in exponential and plateau (G0) phase were analyzed for proliferation associated antigen expression. Expression of PCNA and p145 during G0 was markedly depressed (< 12% positive) while 54% of the G0 cells stained positive for p120. The fluorescent intensity (mean channel fluorescence) of these G0 positive p120 cells, however, was only slightly above the mean channel fluorescence (MCF) of cells stained with a negative isotype control. In asynchronous cultures, all three antigens were expressed in >70% of the cells, with PCNA staining being greater than 95%. Cells were then synchronized using mitotic selection (mitotic index of 97%) and antigen levels were measured as cells progressed synchronously through the cell cycle. From DNA analysis histograms, it appeared that the degree of synchrony was approximately 90% throughout the remainder of the cell cycle. The bivariate DNA/PCNA, DNA/p120, and DNA/p145 histograms for mitotic cells indicated that both p120 and p145 expression were elevated (percent positive and MCF) while PCNA levels were near controls (MCF). In early G1, all three markers were depressed (< 12% positive); however PCNA levels rose precipitously in mid‐G1 (> 50% positive). In late G1 to early S, p145 levels increased concomitantly with increases in p120. All three antigens were elevated throughout S phase and began to decline as cells moved from G2/M to G1 of the next cell cycle with pl45 expression decreasing first. This report indicates that all three proliferation associated antigens studied are differentially expressed in the cell cycle and therefore may be useful in detecting and assessing the proliferation state.
Importance of bicarbonate ion for intracellular pH regulation in antigen‐ and ionomycin‐stimulated RBL‐2H3 mast cells
Authors: Lee Rebecca J. , Oliver Janet M. , Deanin Grace G. , Troup Charles D. , Stump Robert F. 
Published: August 2005

AbstractIn RBL‐2H3 rat basophilic leukemia cells, Ca2+ influx and secretion are activated by antigens that crosslink IgEreceptor complexes and by the Ca2+ ionophore, ionomycin. Here we report that antigen‐stimulated Ca2+ influx and secretion are impaired and ionomycin‐induced responses are strongly inhibited following the removal of HCO3 − from the medium. These results raised the possibility that HCO3 − ‐dependent pH regulation mechanisms play a role in the cascade of events leading to mast cell activation. To test this hypothesis, intracellular pH (pHi) was measured by ratio imaging microscopy in individual RBL‐2H3 cells labeled with 2',7'‐bis(2‐carboxyethyl)‐5‐(6) carboxyfluorescein (BCECF). In unstimulated cells, it was found that basal pHi in the presence of HCO3 − is 7.26, significantly greater than pHi in its absence, 7.09 (P < 10−6). These results, as well as evidence that pHi increases rapidly when HCO3 − is added to cells initially incubated in HCO3 − ‐free medium, indicate that unstimulated cells use a HCO3 − ‐dependent mechanism to maintain cytoplasmic pH. Further analyses comparing unstimulated with stimulated cells showed that antigen causes a small transient acidification in medium containing HCO3 − and a larger sustained acidification in HCO3 − ‐depleted medium. Ionomycin is a more potent acidifying agent, stimulating a sustained acidification in complete medium and causing further acidification in HCO3 − ‐free medium. These results support the hypothesis that the inhibition of antigen‐ and ionomycininduced 45Ca2+ influx and secretion in cells incubated in HCO3 − ‐free medium is at least partially due to the inactivation of HCO3 − ‐dependent mechanisms required to maintain pH in unstimulated cells and to permit pH recovery from stimulus‐induced acidification.
Models for the electronic processing of flow cytometric data at high particle rates
Authors: van Rotterdam A. , Keij J. , Visser J. W. M. 
Published: August 2005

AbstractIn flow cytometry at high particle rates, complete processing of data is limited by the capacity of the flow cytometer electronics which are constrained by the waiting or cycling time of the processor.Four models of impulse processors were analyzed to study the influence of the waiting time and reset mechanisms on the input‐output properties of commonly used electronic devices. The models contain a feedback loop to represent a waiting time and describe reset mechanisms to filter trains of consecutive pulses such as clumps and doublets. Discrete systems analytical tools have been used to derive formulas for the yield of a simple waiting time device, a doublet filter, a clump filter and a clump and doublet filter. Also, the response to a sudden onset of an input signal has been analyzed and is described. The reset mechanism is an important determinant of the capacity of a waiting time device depending on the impulse rate of the input signal.
Comparison of lymphocyte immunophenotyes obtained simultaneously from two different data acquisition and analysis systems on the same flow cytomete,
Authors: Margolick Joseph B. , Scott Elvia R. , Chadwick Karen R. , Shapiro Howard M. , Hetzel A. Dorn , Smith S. Jay , Vogt Robert F. 
Published: August 2005

AbstractImmunophenotyping of different lymphocyte populations was carried out in parallel on 113 consecutively received specimens of human peripheral blood using 2 different data acquisition and analysis systems (EPICS C and 4Cy‐Acmecyte) on the same flow cytometer (EPICS C). The phenotypes analyzed were CD3+, CD4+, CD8+ CD56+ CD16+ CD3−, TCR‐γδ+ CD8−, and TCR‐γδ+ CD8+. Both HIV− and HIV+ specimens were used for this study, including some with CD4 levels as low as 2% of all lymphocytes. Despite differences in gating procedures and shapes of bitmap (rectilinear vs. “amorphous”), the 2 methods agreed to within 2% positive cells in 97% of the cases. Although some statistically significant biases in the methods were observed, these were small and not biologically important. We conclude that both methods of data acquisition and analysis, as employed by experienced operators on the EPICS C flow cytometer, gave essentially equivalent results for lymphocyte subpopulations in peripheral blood preparations.
Flow cytometric analysis of proliferation associated nuclear antigens using washless staining of unfixed cells
Authors: Landberg Göran , Roos Göran 
Published: August 2005

AbstractThe expression of different proliferation associated nuclear antigens was analyzed using a washless double‐staining method and flow cytometry. It is a simple and rapid two‐step procedure which can be performed on love cell numbers. A series of hematopoietic cell lines and fresh lymphoma cells were tested axed the methodology was found to be applicable to a number of nuclear antigens (PCNA, Ki‐67, p105, MPM‐2, fibrillarin). For PCNA, the detectability was dependent on the type of antibody used. The immunofluorescence pattern observed by microscopy was altered for antigens stained by the washless technique in comparison with the pattern obtained with fixed cells. With the washless method, detailed cell cycle analysis could be obtained by dual parameter analysis of PCNA and Ki‐67.
BrdUrd incorporation studies for evaluation of spermatogenesis in the blue fox
Authors: Clausen O. P. F. , Andersen Berg K. , Kirkhus B. , De Angelis P. , Huitfeldt H. 
Published: August 2005

AbstractThe utility of BrdUrd incorporation techniques for studies of spermatogenesis was investigated in the blue fox. BrdUrd was injected intraperitoneally followed by collection of testicular tissue by castration/hemicastration at intervals up to 35 day after pulse labelling. Fluorescent tagged monoclonal antibodies against BrdUrd allowed detection of cells with incorporated tracer in histological sections by fluorescent light microscopy as well as in isolated testicular cells by bivariate BrdUrd/DNA flow cytometry.The duration of the spermatogenic cycle was estimated by following the labelled cohort of preleptotene spermatocytes by immunofluorescence in sections through the various stages of maturation to the late spermatid stage. These data were confirmed by bivariate BrdUrd/DNA flow cytometry of testicular cells in suspensions. Furthermore, estimations of the S phase durations and length of the spermatogonial cell cycle were possible. A consistent and satisfactory fluorescence intensity of incorporated label throughout the study shows that degradation of the incorporated label is no practical problem for this type of study, and suggests that the method is an excellent tool for studying aspects of proliferation and maturation during normal as well as perturbed spermatogenesis.Advantages of the described method include avoidance of potential radiation influence on spermatogenesis from commonly used radiolabelled tracers, e.g., 3H‐TdR, and that both large and small animals can be investigated at modest cost since the unlabelled BrdUrd is considerably less expensive than labelled tracers.
Sequential paraformaldehyde and methanol fixation for simultaneous flow cytometric analysis of DNA, cell surface proteins, and intracellular proteins
Authors: Pollice Agnese A. , Philip Mccoy J. , Shackney Stanley E. , Smith Charles A. , Agarwal Jyotsna , Burnolt Dennis R. , Janocko Laura E. , Hornicek Francis J. , Singh Sarita G. , Hartsock Robert J. 
Published: August 2005

AbstractA cell fixation and permeabilization procedure consisting of sequential paraformaldehyde and methanol was evaluated and found suitable for concomitant flow cytometric quantification of total cellular DNA, immunofluorescence measurements of cell surface proteins, and immunofluorescence measurements of intracellular proteins. Paraformaldehyde/methanol‐fixed cells exhibited significantly greater intracellular antitubulin immunofluorescence than cells fixed with paraformaldehyde or methanol alone (p < 0.002) and significantly greater intracellular antitubulin immunofluorescence than cells fixed with methanol followed by paraformaldehyde (p < 0.006). With paraformaldehyde/methanol fixation, cell morphology was well preserved and forward and right angle light scatter properties were sufficiently well maintained to permit gating on these parameters. Cell surface marker staining with fluorescent anti‐leukocyte antibodies was unaffected by fixation with paraformaldehyde/methanol. Paraformaldehyde effects on the intensity oaf DNA staining with propidium iodide were dependent on paraformaldehyde concentration and Fixation temperature; these effects were least pronounced at low paraformaldehyde concentrations (0.25% or less), and at temperatures lower than 37°C. Paraformaldehyde fixation may result in differences in propidium iodide staining of DNA in some diploid cells, which may produce small spurious aneuploid peaks in normal peripheral blood leukocytes. Paraformaldehyde fixation also produces an apparent increase in the DNA index of aneuploid cell populations in comparison with methanol fixation, particularly when the DNA index exceeds 1.5. Occasionally, this paraformaldehyde fixation‐induced effect is useful in identifying biologically distinct near‐diploid subpopulations in tumors.
Flow cytometric analysis of opposite effects of a monokine on proliferation and differentiation of murine B lymphocytes
Authors: Souvannavong Vongthip , Brown Spencer C. , Adam Arlette 
Published: August 2005

AbstractA 35,000 mw factor able to replace macrophages (FRM) in the induction of the in vitro antibody response to sheep erythrocytes has been isolated from the supernatant of murine resident peritoneal macrophage cultures. Purified FRM, when added at the outset of cultures, induced B cells to generate an antigen‐specific antibody response. The signals provided by FRM in the process of B cell activation were analyzed using a polyclonal model. Cell cycle analysis by multiparameter flow cytometry after acridine orange staining showed that FRM, on its own, did not trigger the transition of B cells from the GO to the G1 stage of the cell cycle. In addition, FRM affected neither the basal intracellular free calcium leve ([Ca2+]i) nor the rapid increase in [Ca2+]i induced by crosslinking of membrane immunoglobulin (mIgM) with anti‐μ, antibodies. In parallel with its positive effect on B cell differentiation, FRM markedly reduced both proliferation and cell cycle progression of B cells stimulated with anti‐μ, plus interleukin 4 (IL‐4). Indeed, the addition of FRM to such cultures led to a preferential accumulation of cells in the early G1 compartment of the cell cycle and to a decreased frequency of cells in all other phases including GIB, S and G2/M.
Aggregation of Escherichia coli B/r A during agar filtration: Effect on morphometric measurements
Authors: Vardi E. , Grover N. B. 
Published: August 2005

AbstractWe have investigated the phenomenon of particle aggregation in a sample of 71,038 Escherichia coli B/r A cells in balanced exponential growth, during preparation for electron microscopy by agar filtration. The bacteria were photographed in a transmission electron microscope and the dimensions and spatial relationships among all the members of each aggregate were recorded using an interactive image processing system. The proportion of aggregated cells, 22%, is much greater than that found by direct count in a light microscope (7%), implying that most aggregation takes place during the preparation stages. The aggregated cells are about 1% narrower than the freecells, because of mutual compression, and 1.5% longer, because of a selection bias in favor of longer cells. From a statistical analysis of the data, we conclude that the clustering of cells into aggregates in the course of sample preparation is the result of random encounters during the settling on the collodion membrane and of the changing surface tension during the drying process. A method is proposed to correct morphometric measurements for the distortion caused by cellular aggregation of this kind.
Preparation and microscopic visualization of multicolor luminescent immunophosphors
Authors: Beverloo H. B. , van Schadewijk A. , Bonnet J. , van der Geest R. , Runia R. , Verwoerd N. P. , Vrolijk J. , Ploem J. S. , Tanke H. J. 
Published: August 2005

AbstractThe preparation of charge‐stabilized suspensions of small phosphor particles (0.1–0.3 μm) and their coupling with antibodies to immunoreactive conjugates is described. Phosphor particles consisting of yttriumoxisulfide activated with europium served as a model system in the evaluation of the stabilizing properties of several polycarboxylic acids. The optimal reagents were then applied to other phosphors which differ in spectral characteristics as well as in luminescence lifetime. These phosphors were ground to a size of 0.1–0.3 μm and proteins or other macromolecules were adsorbed to the phosphor particles to prepare conjugate, of different physico‐chemical properties. A time‐resolved microscope, suitable for real time visualization of the time‐delayed luminescence of the immunophosphors by the human eye, is described in detail. Since most phosphors require excitation with far UV light, a special fluorescence microscope allowing far UV excitation was developed for conventional visualization of the luminescence emitted by the phosphor. The possibility of multiple color labeling using various phosphor conjugates was demonstrated in a model system consisting of haptenized latex beads. © 1992 Wiley‐Liss, Inc.
Image cytometric DNA analysis in human breast cancer analysis may add prognostic information in diploid cases with low S‐Phase fraction by flow cytometry
Authors: Baldetorp Bo , Fernö Mårten , Fallenius Anders , Fallenius‐Vecchi Ghita , Idvall Ingrid , Olsson Håkan , Sigurdsson Helgi , Åkerman Måns , Killander Dick 
Published: August 2005

AbstractMeasurements of DNA ploidy can be performed either with image cytometry (ICM) or flow cytometry (FCM); both methods provide independent prognostic information in primary breast cancer. The aim of the present investigation was to compare the two methods and to relate the findings to prognosis (median followup 42 months). Concordance in ploidy status (diploid, tetraploid, aneuploid) was obtained in 76% of the samples (168/222). When the fraction of S‐phase cells (SPF) from FCM analysis was also taken into consideration, four different groups of samples were obtained (Flow I–IV), which were considered to correspond to the Auer classification (Auer I–IV) of DNA histograms obtained from image cytometry. Complete concordance between the two techniques now was 70% (155/222). Samples classified as Flow I (diploid or near‐diploid with low SPF) and Auer I had a distant metastasis rate of 3/60 (5%), as compared to 62/154 (40%) for all other combinations of the Flow and Auer classifications taken together. Thus, the only findings of prognostic importance were that some samples were Flow I but not Auer I, or vice versa. These two groups represent 17 (7.7%) and 14 (6.3%), respectively, of the total number of samples, and had frequencies of distant metastasis similar to those of the other high‐risk groups, namely, 7/17 and 5/14, respectively. In a multivariate analysis, flow cytometric S‐phase value was a stronger prognostic factor than either the Flow and Auer classification.We conclude that when routine FCM DNA analysis is used, diploid or neardiploid samples with a low S‐phase value should be reanalyzed with ICM. © 1992 Wiley‐Liss, Inc.