bluuebunny/crossref_metadata_2025 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

Mammalian 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

Although 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

The 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

Flow 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

In 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

In 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

Immunophenotyping 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

The 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

The 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

A 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

A 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

We 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

The 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

Measurements 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.
Neutrophil function screenlng in patients with chronic granulomatous disease by a flow cytometric method
Authors: Epling C. L. , Stites D. P. , McHugh T. M. , Chong H. O. , Blackwood L. L. , Wara D. W.
Published: August 2005

Neutrophils from patients with chronic granulomatous disease (CGD) fail to produce a significant oxidative burst following stimulation. We have evaluated the use of flow cytometry and the dye 2′,7′dichlorofluorescein diacetate (DCF) for routine screening for deficiencies of neutrophil oxidative burst. A range for DCF fluorescence for phorbol myristate acetate stimulated and non‐stimulated neutrophils was established based on data from 52 healthy adults. Samples from three patients with suspected neutrophil dysfunction, three patients with X‐linked CGD, and one patient with autosomal recessive (AR) CGD were evaluated with both the DCF assay and the quantitative nitroblue tetrazolium dye reduction (NBT) test. For the DCF test, the ratio of mean fluorescence intensity of stimulated to non‐stimulated neutrophils was <5 for CGD patients and from 16 to >50 for healthy individuals. With the DCF test, two populations of neutrophils could be identified in samples from four carriers of X‐linked CGD, although two carriers of AR CGD had NBT and DCF results in the normal range. Our data suggest the DCF test is a sensitive and convenient method for detecting CGD. © 1992 Wiley‐Liss, Inc.
Drop‐delay measurement using enzyme‐coated particles
Authors: Lazebnik Y. A. , Poletaev A. I. , Zenin V. V.
Published: August 2005

A simple technique is suggested for the measurement of drop delay for flow sorting. While the flow cytometer was set to sort a fixed number of particles, the drop‐ delay setting was changed step by step, and at each step the HRP‐coupled particles were sorted into a well of an immunoassay strip. Then the HRP activity of the sorted samples was revealed by routine methods. The maximum level of the enzyme activity shows the proper drop‐delay setting. Determination of the drop‐delay setting takes only a few minutes. The technique is independent of the type of flow cytometer and does not require any additional equipment. © 1992 Wiley‐Liss, Inc.
Protooncogene expression in subpopulations of cells front leukemia patients
Authors: Hulette Ben C. , Banavali Shripad D. , Finke Daniel P. , Gopal Venu , Preisler Harvey D.
Published: August 2005

This report describes a method for pre‐serving the light scatter patterns of cells in which myc and myb expression are being measured. Exposure of cells to 1% paraformaldehyde for 72 h prior to antibody staining for myc and myb proteins preserved the light scatter patterns. Using this method, myc and myb expression was found to be highest in lymphocytes and monocytes and lowest in granulo cytes. The measurement of differences in the level of expression of these genes in subpopulations of leukemia cells obtained from individual patients is possible as is assessment of the levels of expression amongst normal and leukemia cells present in the same patient. © 1992 Wiley‐Liss, Inc.
Photo‐bleaching and photon saturation in flow cytometry
Authors: van den Engh Ger , Farmer Colleen
Published: August 2005

In flow cytometry, small particles travel at a high speed through a bright light spot. The high light intensity at the point of measurement causes measurable photon saturation. This observation indicates that the rate at which individual dye molecules emit photons is close to the maximum emission rate. Despite the short exposure time, individual molecules may go through a few hundred excitation cycles while they are in the light beam. The absorbed light dose causes significant dye destruction. This article presents experimental procedures to determine the extent of photon saturation and photo‐bleaching of dyes bound to cell nuclei in a flow cytometer. Measurements of Hoechst and propidium iodide bound to chromatin show that the amount of dye bleached per emitted photon is the same at low and high illumination intensities. This finding indicates that photon emission and dye destruction are both the result of the absorption of single excitation photons. The experimental observations allow rough estimates of the lifetime of the excited state and the lifetime of the molecule. The lifetime of the Hoechst 33258 bound to DNA is estimated to be 100 excitation‐relaxation cycles. The average propidium iodide molecule lasts ∼ 200 excitation‐relaxation cycles. The theoretical considerations show that the optimal illumination conditions are different for bleaching and nonbleaching dyes. Aoa optical arrangement for high precision measurements of bleaching dyes is presented. Published 1992 by Wiley‐Liss, Inc.
Signal transduction in monocytes and granulocytes measured by multiparameter flow cytometry
Authors: Lund‐Johansen Fridtjof , Olweus Johanna
Published: August 2005

The novel calcium indicator fura red and the oxidative burst indicator dihydrorhodamine (both excited at 488 nm) were used in combination with multiparameter flow cytometry to allow simultaneous kinetic measurements of calcium fluxes and oxidative bursts in monocytes and granulocytes. Using this method it was possible to obtain direct evidence for the following cell type‐ and stimulus‐specific differences in signal transduction pathways: (1) n‐formyl‐methionyl‐leucylphenylalanine (FMLP)/cytochalasin B‐induced oxidative burst is several‐fold higher in granulocytes than in monocytes although the calcium fluxes have similar amplitudes in the two cell types; (2) stimulus‐induced calcium fluxes in granulocytes are mainly due to release from intracellular stores, whereas monocytes mobilize calcium mainly by influx from the medium; (3) the FMLP/cytochalasin Binduced calcium flux in monocytes is less sensitive to the G‐protein inhibitor pertussis toxin than the flux in granulocytes; (4) in contrast to FMLP/cytochalasin B, the protein kinase C activator phorbol myristate acetate (PMA) induces an oxidative burst that is not preceded by a cytoplasmic calcium flux; (5) the PMAinduced oxidative burst can be triggered in monocytes and granulocytes that are depleted of intracellular calcium ions, whereas that induced by FMLP/cytochalasin B can not; (6) the G‐protein inhibitor pertussis toxin blocks an early event in the signal transduction pathway of FMLP/cytochalasin B, as shown by inhibition of both calcium fluxes and oxidative burst; and (7) 100 nM of the protein kinase inhibitor staurosporine blocks the FMLP/cytochalasin B‐induced respiratory burst by interfering with a step downstream to cytoplasmic calcium fluxes, whereas only 10–20 nM is necessary to block PMA‐induced oxidative burst. © 1992 Wiley‐Liss, Inc.
Swine chromosomal DNA quantification by bivariate flow karyotyping and karyotype interpretation
Authors: Schmitz Annette , Chaput Brigitte , Fouchet Pierre , Guilly Marie Noelle , Frelat Gerard , Vaiman Marcel
Published: August 2005

Human and swine chromosomes were analyzed separately and as a mix to obtain bivariate flow karyotypes. They were normalized to each other in order to use the human chromosomal DNA content as standard. Our results led to the characterization of the “DNA line” in swine identical to the human “DNA line.” Estimation of the DNA content in megabase pairs of the swine chromosomes is proposed. Chromosomal assignment to the various resolved peaks on the bivariate swine flow karyotype is suggested from the relation between DNA content quantified by flow cytometry and chromosomal size. Swine chromosomes 1, 13, 6, 5, 10, 16, 11, 18, and Y were assigned to peaks A, B, C, K, L, N, O, Q, and Y, respectively. Peaks D anal E were assumed to contain chromosomes 2 and 14, but without specific assignment. Similarly, P and M peaks were expected to correspond to chromosomes 12 and 17. Of the remaining chromosomes (3, 7, X, 8, 15, 9, and 4), chromosomes 3, 7, and X, which were assigned previously to peaks F, G, and H, respectively, led us to deduce that chromosomes 15 and 8 belonged to peaks I and J, and chromosomes 9, 4, and X to peak H. © 1992 Wiley‐Liss, Inc.
Comparative evaluation of fresh, fixed, and cryopreserved solid tumor cells for reliable flow cytometry of DNA and tumor associated antigen
Authors: van Dam P. A. , Watson J. V. , Lowe D. G. , Chard T. , Shepherd J. H.
Published: August 2005

Five different protocols for the shortterm preservation of cells used for multiparameter flow cytometric assay of tumour associated antigens (TAA) and DNA were assessed in cell suspensions prepared by mechanical disaggregation of 15 gynecological tumors. The protocols at 4°C were (1) storage in buffer, (2) storage in 50% methanol, and (3) storage in buffer after formalin fixation. Tissues were also cryopreserved as cell suspensions and tissue blocks. When the TAA expression and DNA histograms of the preserved cells were compared with those in fresh cell suspensions, cryopreservation was found to be the best method: TAA expression was well preserved and there was a good correlation between TAA expression and the quality of the DNA histograms, respectively, in fresh and cryopreserved cells (RS: 0.82–0.91, P<0.001 for all TAAs). The cell suspensions preserved at 4°C all showed a significant increase in background fluorescence (P<0.05) and a reduction in the TAA specific fluorescence (P<0.011). Methanol fixation was better than buffered formalin for the proteins studied, though both gave significantly worse results than cryopreservation. The quality of these cell suspensions and the correlation with TAA measurements in fresh cell suspensions deteriorated progressively with time, particularly if they were stored more than a week. © 1992 Wiley‐Liss, Inc.
Flow‐cytometric enumeration of micronucleated polychromatic erythrocytes in mouse peripheral blood
Authors: Grawé Jan , Zetterberg Gösta , Amnéus Herman
Published: August 2005

A flow‐cytometric assay is described that can be used to determine the frequency and the DNA content of mirronucleated polychromatic (PCE) and normochromatic (NCE) erythrocytes in mouse peripheral blood. Thiazole orange was used for discrimination between PCEs and NCEs, while Hoechst 33342 was used to detect micronucleated PCEs and NCEs. Up to 70,000 polychromatic erythrocytes can be analyzed in less than 10 min. This corresponds to 150–3,000 micronucleated polychromatic erythrocytes, 90–95% of which are true events as determined with a fluorescence microscope after sorting. Using X‐rays as the inducing agent in dose‐response experiments, a significant increase can be registered at doses of 0. 02 Gy. It seems possible that the method will also allow the detection of clastogenic effects of other inducing agents at lower doses than previously possible. © 1992 Wiley‐Liss, Inc.
Three‐dimensional image processing for morphometric analysis of epithelium sections
Authors: Albert René , Schindewolf Thomas , Baumann Irith , Harms Harry
Published: August 2005

The reproducible classification of poorly differentiated abnormal epithelium specimens is still a diagnostic problem. The computer‐aided method described here improves the differentiation between benign and malignant epithelium specimens. Hematoxylin and eosinstained sections of normal squamous epithelium, dysplasia, carcinoma in situ, and carcinoma were scanned in a TV microscope system and analyzed by means of image processing methods on a DEC 5000/200 workstation. From the 15–20 wm thick histological sections, 3–5 focus positions in steps of 1–4 μm were scanned. The segmentation of the cell nuclei was performed automatically by color analysis and geometric operations. For each nucleus the best focus level was selected and at this level the center of the cell was calculated. Graph theoretical methods were applied to analyze the morphometry of the epithelium specimens. The minimal spanning tree was computed in the three‐dimensional (3D) space of the sections with the selected centers of the nuclei as vertices. The best feature found for discrimination of the specimens is the average length of all edges in a tree. In the two‐dimensional (2D) analysis we had to accept an error probability of about 20% in differentiation of dysplasia and carcinoma. In contrast to this we differentiated normal squamous epithelium, dysplasia, and carcinoma with a correct classification rate of 100% in the 3D analysis. © 1992 Wiley‐Liss, Inc.
Simultaneous analysis of immunophenotype and apoptosis of murine thymocytes by single laser flow cytometry
Authors: Bruce Lyons A. , Samuel Kay , Sanderson Andrew , Maddy Alun H.
Published: August 2005

The study of the role of apoptosis in thymocyte development has been hampered by the lack of a means of directly immunophenotyping cells undergoing the early phase of apoptosis. This restriction has been overcome by single laser flow cytometry in which apoptosis is detected by Ethidium Bromide (EBr) staining and cell phenotype by binding of FITC‐labelled antibody. The initial phase of apoptosis is observed as a cell population that stains faintly with EBr preceding the characteristically bright EBrstaining normally associated with cell death. Here we directly demonstrate using single laser flow cytometry that CD4+ CD8+ CD3low/CD3ntermediate thymocytes undergo apoptosis in vitro in response to glucocorticoid treatment. © 1992 Wiley‐Liss, Inc.
Quantification of inter‐ and intra‐nuclear variation of fluorescence in situ hybridization signals
Authors: Nederlof P. M. , van der Flier S. , Raap A. K. , Tanke H. J.
Published: August 2005

This study aims at the quantification of specific DNA sequences by using fluorescence in situ hybridization (ISH) and digital imaging microscopy. The cytochemical and cytometric aspects of a quantitative ISH procedure were investigated, using human peripheral blood lymphocyte interphase nuclei and probes detecting high copy munber target sequences as a model system. These chromosome‐specific probes were labeled with biotin, digogigenin, or fluorescein

Quantification of the fluorescence ISH signals was performed using an epifluorescence microscope equipped with a multi‐wavelength illuminator, and a cooled charge coupled device (CCD) camera. Specific image analysis programs were developed for the segmentation and analysis of the images provided by ISH

The fluorescence intensity distributions of the ISH spots showed large internuclear variation (CVs up to 65%) for the probes used. The variation in intensity was found to be independent of the probe, the type of labeling, and the type of immunocytochemical detection used. Variation in intensity was not caused primarily by the immunocytochemical detection method, since directly fluorescein‐labeled probes showed similar internuclear variation. Furthermore, it was found that different white blood cell types, which harbor different degrees of compactness of the nuclear chromatin, showed the same variation

The infra‐nuclear variation in intensity of the ISH spots on the two chromosome homologs within one nucleus was significantly smaller (approximately 20%) than the inter‐nuclear variation, probably due to more constant local hybridization conditions. Due to the relatively small intranuclear variation, copy number polymorphisms of the satellite DNA sequence on chromosome 1 could readily be quantified. Furthermore, an internal standard to correct for differences in hybridization efficiency among cells, based on a second hybridization, was introduced

It is concluded that development of an accurate quantitative ISH procedure requires further reduction of the internuclear variation. Results presented indicate that improvements in the preparation of the target material and in the hybridization reaction are especially needed to fulfill this requirement© 1992 Wiley‐Liss, Inc.
Flow cytometric determination of atypical antigen expression in acute leukemia for the study of minimal residual disease
Authors: Drach Johannes , Drach Doris , Glassl Herta , Gattringer Claus , Huber Heinz
Published: August 2005

The aim of this study was to investigate to which extent acute leukemias could be monitored for residual disease by using atypical antigen combinations as leukemia‐related markers. Atypical antigenic features were determined by double color flow cytometry and included coexpression of lymphoid and myeloid related antigens, unnphysiological coexpression of immature and mature antigens, and lack of an antigen that is normally expressed during maturation. Atypical immunophenotypes were detected in 35 of 68 patients with AML (51.5%) and 15 of 24 patients with ALL (62.5%). When 12 patients with leukemia‐associated markers were again analyzed at relapse, the relevant antigen combinations were retained in 11 of them. The sensitivity of this two color flow cytometric assay as determined in dilution experiments was 1 in 103 to 104 cells

Follow‐up studies of bone marrow samples revealed that, after induction chemotherapy cells with leukemia‐associated markers were detectable in several patients at a frequency of 0.5 to 4%, but only patients in whom the cells with atypical antigens never disappeared suffered from relapse. In contrast, patients who became negative for the atypical cells remained in complete remission (median remission duration after the first negative bone marrow assessment by flow cytometry 52 weeks, range 20–102)

We conclude that atypical antigen combinations, which are present in a meaningful number of acute leukemias, are a valuable means of monitoring acute leukemia patients during follow‐up. This flow cytometric approach can complement other strategies to get a more accurate definition of remission in acute leukemia. © 1992 Wiley‐Liss, Inc.
Flow cytometric analysis of altered mononuclear cell transmembrane potential induced by cyclosporin
Authors: Tanner Michael K. , Wellhausen Samuel R. , Klein Jon B.
Published: August 2005

Immunosuppression by the fungal metabolite cyclosporin A (CsA) is characterized by functional inhibition, rather than destruction of cells. Because activation of immune cells involves intracellular signalling events associated with modulations of cell transmembrane potential (TMP), we tested the ability of cyclosporin A (CsA) to modulate immune mononuclear cell TMP in vitro using a TMP sensitive cationic dye, dihexyloxacarbocyanine (DIOC6(3)). All analyses were performed by flow cytometry. CsA increased TMP in monocytes and lymphocytes isolated from the blood of healthy human volunteers. CsA‐induced hyperpolarization was time and concentration dependent in monocytes while the lymphocyte hyperpolarization, although time dependent, was evident over the entire range of CsA concentrations tested. CsA‐induced hyperpolarization of lymphocytes was dependent on potassium ion (K+) efflux as indicated by the absence of hyperpolarization in 154 mM KCl or with pretreatment with 100 üMM quinine (an inhibitor of K+ channels). Monocyte hyperpolarization by CsA was not inhibited in either system. Dihydrocyclosporin C (DH‐CsC), an immunosuppressive analog of CsA, also hyperpolarized mononuclear cells. The anionic TMP sensitive dye bis oxonol (di‐BA‐C4) indicated that CsA treatment depolarized mononuclear cell plasma membranes. The mitochondrial poison carbonyl cyanide p‐trifluoromethoxy‐phenyl‐hydrazine (FCCP) eliminated CsA induced hyperpolarization and also indicated that CsA caused plasma membrane depolarization. We conclude that brief in vitro exposure to cyclosporin alters the transmembrane electrical potential of human lymphocytes and monocytes. © 1993 Wiley‐Liss, Inc.
Micromanipulation by “multiple” optical traps created by a single fast scanning trap integrated with the bilateral confocal scanning laser microscope
Authors: Visscher K. , Brakenhoff G. J. , Krol J. J.
Published: August 2005

We have developed a novel micromanipulator consisting of multiple optical traps created by scanning one single beam trap along a variable number of positions. Among other things, this enables the orientation of irregularly shaped and relatively large structures which could not be oriented by just one trap as is demonstrated on long Escherichia coli bacteria filaments. We expect that the multiple trap manipulator will broaden the field of applications of optical trapping as a micromanipulation technique. For example, it facilitates the study of mechanical properties of extended structures as illustrated by a “bending”‐experiment using E. coli bacterium filaments. A special application of the multiple trap manipulator is the “indirect trapping” of objects which we did by keeping them held between other optically trapped particles. Indirect trapping makes it possible to trap particles which either cannot be trapped directly due to their optical properties (refractive index) or for which exposure to the laser radiation is undesirable. The multiple optical trap manipulator is controlled interactively by a UNIX workstation coupled to a VME instrumentation bus. This provides great flexibility in the control of the position and the orientation of the optical traps.

Micromanipulation makes it desirable to have real time 3D microscopy for imaging and guidance of the optical traps. Therefore we integrated optical micromanipulation and a specially developed real‐time confocal microscope. This so called bilateral confocal scanning laser microscope (bilateral CSLM) [Brakenhoff and Visscher, J Microsc 165:139–146, 1992] produces images at video rate. © 1993 Wiley‐Liss, Inc.
Optimizing fluorescent in situ hybridization with rRNA‐targeted oligonucleotide probes for flow cytometric identification of microorganisms
Authors: Wallner Günter , Amann Rudolf , Beisker Wolfgang
Published: August 2005

A combination of fluorescent rRNA‐targeted oligonucleotide probes (“phylogenetic stains”) and flow cytometry was used for a high resolution automated analysis of mixed microbial populations. Fixed cells of bacteria and yeasts were hybridized in suspension with fluorescein‐ or tetramethylrhodamine‐labeled oligonucleotide probes complementary to group‐specific regions of the 16S ribosomal RNA (rRNA) molecules. Quantifying probe‐conferred cell fluorescence by flow cytometry, we could discriminate between target and nontarget cell populations. We critically examined changes of the hybridization conditions, kinetics of the hybridization, and posthybridization treatments. Intermediate probe concentrations, addition of detergent to the hybridization buffer, and a posthybridization washing step were found to increase the signal to noise ratio. We could demonstrate a linear correlation between growth rate and probe‐conferred fluorescence of Escherichia coli and Pseudomonas cepacia cells. Oligonucleotides labeled with multiple fluorochromes showed elevated levels of non‐specific binding and therefore could not be used to lower the detection limits, which still restrict studies with fluorescing rRNA‐targeted oligonucleotide probes to well‐growing microbial cells. Two probes of different specificities—one labeled with fluorescein, the other with tetramethylrhodamine—could be applied simultaneously for dual color analysis. © 1993 Wiley‐Liss, Inc.
Statistical confirmation that immunofluorescent detection of DNA repair in human fibroblasts by measurement of bromodeoxyuridine incorporation is stoichiometric and sensitive
Authors: Selden Jules R. , Dolbeare Frank , Clair James H. , Nichols Warren W. , Miller Judith E. , Kleemeyer Katherine M. , Hyland Robert J. , DeLuca John G.
Published: August 2005

Diploid human fibroblasts (IMR‐90 cells), grown to confluency and growth‐arrested by serum starvation, were irradiated with a variety of doses of UV light (0.025–40 J/m2) or incubated with broad dose ranges of four direct‐acting mutagens [ethyl methanesulfonate (EMS), ICR‐170, methyl methanesulfonate (MMS), and 4‐nitroquinoline oxide (4‐NQO)] and pulsed with a thymidine analog, 5‐bromodeoxyuridine (BrdUrd) to detect evidence of DNA repair. These cells and appropriate controls were immunochemically stained and subjected to flow cytometric analysis to quantify BrdUrd incorporation into DNA and simultaneously measure cellular DNA content. Since the maximal quantity of BrdUrd incorporated with repairing cells is profoundly less than the amount incorporated during replicative synthesis and flow cytometric analysis collects information on a cell‐to‐cell basis, data collection using linear histograms succeeded both in revealing repairing cellular populations and eliminating replicative cells from the analysis. Technical modifications necessary to achieve stoichiometry with UV‐irradiated IMR‐90 fibroblasts included a 48h repair (and pulse) period, followed by denaturing cellular DNA for 15 min at 90°C. The limit of detection was equal to or below the lowest dose investigated (0.025 J/m2). DNA repair was also detected with cultures incubated with low doses of all chemicals and pulsed with BrdUrd for a 24 h period. The limits of detection were near or below 500 m̈M EMS, 5 m̈M MMS, 0.25 m̈M 4‐NQO, and 0.1 m̈M ICR‐170. © 1993 Wiley‐Liss, Inc.