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W Beisker

Publications and source records attributed to W Beisker.

35 records · Page 2Linked to original sources
PubMed1993

Flow cytometric analysis of bromodeoxyuridine-induced micronuclei.

The effects of DNA substitution by the thymidine analogue 5-bromodeoxyuridine (BrdU) on cell cycle progression and micronucleus induction were studied in different mammalian cell cultures. Simultaneous flow cytometric measurements of DNA content and side scatter of nuclei in Chinese hamster embryo (CHE) cells revealed a concentration-dependent temporary block in the G2/M phase of the first cell cycle. NIH 3T3 cells and human amniotic fluid fibroblast-like cells, on the contrary, did not show any cell cycle disturbances in the presence of BrdU. Micronucleus frequency increased as soon as CHE cells started to divide and reached a plateau when all cells have divided. The height of this plateau was almost equal for 60 and 100 microM BrdU. This saturation of micronucleus induction was due to a saturation of BrdU incorporation into DNA already at a doses of 60 microM as shown by the BrdU/Hoechst quenching technique. Indirect immunofluorescent staining of kinetochores with CREST antibodies revealed that nearly all BrdU-induced micronuclei were kinetochore-negative suggesting the presence of acentric chromosome fragments in these micronuclei. DNA distributions of micronuclei measured by flow cytometry showed several peaks representing micronuclei which contain DNA fragments of defined sizes induced by non-random breakage of chromosomes 1 and X as verified by flow karyotyping and C-banding.

3T3 Cells↗
PubMed1993

Flow cytometric detection of micronuclei induced by chemicals in poly- and normochromatic erythrocytes of mouse peripheral blood.

In order to standardize automated scoring for the in vivo micronucleus (MN) test a flow cytometric method which recognized micronucleated polychromatic erythrocytes (MPCE) and micronucleated normochromatic erythrocytes (MNCE) in mouse peripheral blood developed by Grawé et al. (1992) has been modified and applied. Blood samples were purified with 35% percoll solution and stained with the RNA-specific dye thiazole orange (TO) and with the DNA-specific dye Hoechst 33342 (HO) for dual laser flow cytometry. The TO fluorescent signals permitted the discrimination between polychromatic and normochromatic erythrocytes (PCE and NCE). Cytotoxic effects could be assessed by the reduction of PCE counts. The blue fluorescent signals of HO permitted the scoring of MN. The MPCE and MNCE were flow sorted for microscopic analysis and showed that 95% of the sorted cells actually contained MN. Three model chemicals, the clastogen mitomycin C, the aneugen colchicine and the industrial chemical acrylamide were tested at 24 h intervals after single intraperitoneal injection up to 72 h after treatment of male (102/ElxC3H/El)F1 mice. All three chemicals showed a dose-related maximum of the MPCE frequencies at 48 h while the MNCE frequencies stayed within the control range up to 72 h. The data obtained with the flow cytometric method were in good agreement with published results. The flow cytometric technique presented here is a fast, accurate and automated method for quantifying MPCE and MNCE in peripheral blood as an indicator of cytogenetic damage induced in the bone marrow and scored in peripheral blood samples. With minor modifications the technique will also be applicable to bone marrow samples.

Acrylamide↗
PubMed1992

Multiparametric flow cytometric analysis of radiation-induced micronuclei in mammalian cell cultures.

A new flow cytometric method is presented that quantifies the frequency of radiation-induced micronuclei in mammalian cell cultures with high precision. After preparing a suspension of main nuclei and micronuclei stained with ethidium bromide and Hoechst 33258, both types of particles are measured simultaneously in a flow cytometer using forward light scatter and three fluorescence emission intensities excited by UV, 488 nm, and by energy transfer from Hoechst 33258 to ethidium bromide. Nonspecific debris overlapping the micronucleus distribution especially in the low fluorescence intensity region was discriminated from micronuclei by calculating ratios of the different fluorescences. The frequencies of radiation-induced micronuclei measured with this new technique agreed well with results obtained by conventional microscopy. The lower limit of the DNA content of micronuclei identified by this technique was found to be about 0.5%-0.75% of the DNA content of G1-phase nuclei. Dose effect curves and the time-dependent induction of micronuclei were measured for two different mouse cell lines.

Animals↗
PubMed1992

An automated flow cytometric micronucleus assay for human lymphocytes.

A new flow cytometric method is presented for scoring micronuclei (MN) in human lymphocytes after in vitro gamma-irradiation. Fifty to fifty-five hours after PHA-stimulation, the frequency of micronuclei per nucleus and the fraction of cells in the second cell cycle were measured using flow cytometry. All data were automatically analysed using our DAS-software package. Eight individual linear-quadratic dose response curves derived from five donors revealed inter- and intra-individual variabilities of all curve parameters. Since also an age dependence was found for spontaneous MN-frequencies and for the linear curve parameter, a combined linear-quadratic age-dose-effect model was used to fit the data. The 90% prediction intervals show that a reliable individual dose estimation for donors aged between 23 and 54 years cannot be achieved for exposures below 1 Gy.

Adult↗
PubMed1992

Micronuclei induced by 2-chlorobenzylidene malonitrile contain single chromosomes as demonstrated by the combined use of flow cytometry and immunofluorescent staining with anti-kinetochore antibodies.

The effects of the tear gas 2-chlorobenzylidene malonitrile (CS) on micronucleus induction and cell cycle kinetics were studied in Chinese hamster and Ehrlich ascites tumour cells using flow cytometric analysis of micronuclei and nuclei in suspension, and indirect immunofluorescent staining of kinetochores in micronuclei. In both cell lines CS induced a concentration-dependent inhibition of the fraction of cells in mitosis as observed by simultaneous flow cytometric measurements of DNA content and side scatter intensities of cell nuclei. Micronucleus frequency increased during the delayed division of cells accumulated by CS in mitosis and reached a plateau when most of these cells have divided. The height of this plateau depended on the CS concentration. Results obtained by flow cytometric analysis of the frequency of CS-induced micronuclei did not agree quantitatively with results obtained by microscopic analysis due to cells showing CS-induced fragmented nuclei. Nearly all CS-induced micronuclei exhibited kinetochores, the majority of which (60-70%) showed one kinetochore per micronucleus implying the presence of a single metaphase chromosome in these micronuclei. DNA distributions of micronuclei measured by flow cytometry showed pronounced peaks corresponding to the DNA distribution of chromosomes measured by flow karyotyping. Even micronuclei containing two of the large chromosomes could be observed as distinct peaks in the distributions. The combined results of flow cytometric analysis of micronucleus distributions and immunofluorescence staining of kinetochores in micronuclei suggest that CS induces micronuclei mainly by damaging the spindle fibres of single chromosomes during mitosis, thus possibly leading to aneuploidy and polyploidy.

Animals↗
PubMed1992

Intraphagolysosomal pH in canine and rat alveolar macrophages: flow cytometric measurements.

Intracellular dissolution of inhaled inorganic particles is an important clearance mechanism of the lung and occurs in phagolysosomal vacuoles of phagocytes. Flow cytometric measurements of intraphagolysosomal pH in alveolar macrophages (AM) obtained from beagle dogs, Wistar rats, and from a baboon were made using fluorescein isothiocyanate-labeled amorphous silica particles (FSP). AM were obtained by bronchoalveolar lavage. FSP were phagocytized by AM in cell suspensions incubated in full media for 24 hr up to 6 days. Dual laser flow cytometry was performed and six-parameter list mode data were recorded from forward scatter, side scatter, and fluorescence intensities at 530 nm excited at 457 nm and 488 nm as well as logarithmic fluorescence intensity at wavelengths 630 nm excited at 488 nm. In this way it was possible to discriminate viable AM with phagocytized FSP from lysing AM with phagocytized FSP and from cells without FSP and from free FSP. Viable cells were distinguished from lysing cells by staining with propidium iodide immediately before the flow cytometric measurement. A calibration curve for the pH value was determined from FSP suspended in buffered media at pH values ranging from 3.5 to 7.5. First flow cytometrical results indicated that after an incubation time of 24 hr, the mean intraphagolysosomal pH of viable AM was 4.7 +/- 0.3 for dogs and 5.1 +/- 0.5 for rats. The intraphagolysosomal pH of the baboon AM was 4.5.

Animals↗
PubMed1990

Flow cytometric analysis of G1- and G2/M-phase subpopulations in mammalian cell nuclei using side scatter and DNA content measurements.

Several subcompartments of the cell cycle in addition to the G1-, S-, and G2-phases usually observed were identified by simultaneous flow cytometric measurements of ethidium bromide fluorescence and side scatter intensity of cell nuclei. Metaphase cells and very early G1-phase cells (G1A) with low side scatter intensities were discriminated from interphase cells with high side scatter intensities. The reason for the various side scatter intensities was found to be the different structure of metaphase cells and early G1-phase cells due to chromatin condensation as shown by sorting of the respective cell nuclei. The G1A-phase could further be subdivided into two compartments with very low side scatter (G1A1) and intermediate side scatter (G1A2) intensities. Using partially synchronized cells the duration of these subcompartments of the G1-phase could be estimated. The durations of G1A1- and G1A2-phases were found to be about 10 min and 20 min, respectively, compared to the total duration of the G1-phase of about 3 h. Additional flow cytometric measurements of side scatter intensities of cell nuclei provide therefore further information on subcompartments of the G1- and G2/M-phases.

Animals↗
PubMed1989

Denaturation and condensation of intracellular nucleic acids monitored by fluorescence depolarization of intercalating dyes in individual cells.

The intercalating binding of planar aromatic dye molecules to nucleic acids can be analyzed using fluorescence depolarization measurements of the dye molecules excited by linearly polarized light. In this study, we investigated the conformational changes of the intracellular DNA-dye complex in single cells. Flow cytometry, combined with a newly developed double-beam autocompensation technique, permitted rapid high-precision fluorescence depolarization measurements on a large number of individual cells. The dyes ethidium bromide (EB), propidium iodide (PI), and acridine orange (AO) were used in this study. Depending on the dye-to-phosphate ratio of the nuclear acid-dye complex, as well as on the spatial dye structure itself, internal and external binding sites can be monitored by fluorescence depolarization analysis. Both energy transfer and rotation and vibration of the dye molecules cause depolarization of the fluorescence emission. Differences in the concentration-dependent dye fluorescence depolarization values between PI and EB on one side and AO on the other side can be interpreted as a denaturation and condensation of double-stranded DNA regions by AO. We further show that the fluorescence polarization measurement technique can be used in an alternative way to monitor thermal denaturation of cellular DNA.

Acridine Orange↗
PubMed1988

Measurement of the kinetics of DNA repair synthesis after uv irradiation using immunochemical staining of incorporated 5-bromo-2'-deoxyuridine and flow cytometry.

The kinetics of unscheduled DNA synthesis in normal human fibroblasts was characterized by flow cytometry utilizing the immunofluorescent detection of 5-bromo-2'-deoxyuridine (BrdUrd) incorporated into cellular DNA during the repair process. Quiescent normal human fibroblasts were irradiated with ultraviolet light and incubated in the presence of BrdUrd during a postirradiation repair period. The amount of unscheduled DNA synthesis was then quantified in the quiescent cells by immunofluorescence staining using monoclonal antibodies against BrdUrd incorporated into the DNA. Significant amounts of unscheduled DNA synthesis were measured after doses as low as 0.1 J/m2 and for time periods as short as 15 min. The initial repair rate was found to be linear with time at all doses tested until repair neared completion. Interestingly, the initial repair rate was constant for doses over the range of 5 to 40 J/m2, whereas the time to completion of repair was dose dependent. These results suggest that above 5 J/m2 in normal human fibroblasts, the repair process is saturated but continues to function until all available regions are repaired. Using this methodology for measuring unscheduled DNA synthesis in combination with second and third flow markers, it is now possible to measure unscheduled DNA synthesis in heterogeneous mixtures of cells.

Bromodeoxyuridine↗
PubMed1987

An improved immunocytochemical procedure for high-sensitivity detection of incorporated bromodeoxyuridine.

This report describes an improved immunochemical procedure to stain cells in suspension for incorporated bromodeoxyuridine (BrdUrd) and total DNA content. The procedure consists of five steps: chromatin proteins are extracted by treating with 0.1 M HCl and 0.7% Triton X-100 to facilitate DNA denaturation and to minimize nonspecific staining; cellular DNA is denatured by heating to 100 degrees C in distilled water; BrdUrd in single-stranded DNA (ssDNA) is stained using an immunochemical procedure; autofluorescence is reduced using sodium borohydride (NaBH4); and DNA is stained with the fluorescent dye propidium iodide. With this procedure, the BrdUrd incorporated by CHO cells during periods as short as a few seconds can be detected using flow cytometry. In addition, the stoichiometry of the immunofluorescent staining procedure is high.

Animals↗
PubMed1986

Cell cycle analysis using flow cytometry.

This manuscript reviews the utility of flow cytometry for the study of cell proliferation. The applications of univariate DNA distribution analysis to cytokinetic studies of asynchronous and perturbed cell populations are discussed briefly. The newly developed technique for simultaneous flow cytometric measurement of cellular DNA content and amount of incorporated bromodeoxyuridine is discussed in more detail. The cytochemistry required for this analysis is reviewed as are its applications to: determination of the fractions of cells in the G1-, S- and G2 + M-phases of the cell cycle; determination of the G1-, S- and G2 + M phase durations and dispersions and growth fraction for asynchronous cells; detection of ara-C resistant cells present at low frequency in an otherwise sensitive population; and analysis of the cytokinetic response of a solid murine tumour to treatment in vivo with a cell cycle specific agent.

Animals↗
PubMed1985

Cytochemistry for bromodeoxyuridine/DNA analysis: stoichiometry and sensitivity.

This report describes an improved immunochemical procedure for staining cells in suspension for amount of incorporated bromodeoxyuridine (BrdUrd) and total DNA. In this procedure, cellular DNA is partially denatured by extracting the cells with 0.1 M HCl and then heating them to 80 degrees C in a 50% formamide solution. The cells are then immunofluorescently stained using a monoclonal antibody against BrdUrd in single-strand DNA (ssDNA) and counterstained for DNA content with propidium iodide (PI), a dye that fluoresces preferentially when bound to double-strand DNA (dsDNA). We show that the relative amounts of immunofluorescently stained BrdUrd in ssDNA and PI in dsDNA can be altered reciprocally by changing the formamide concentration, denaturation time, and denaturation temperature. We show that this new immunochemical staining procedure allows more complete DNA denaturation so that fivefold lower levels of BrdUrd incorporation can be quantified. In addition, we show that the BrdUrd-linked immunofluorescence achieved using the new denaturation procedure is more linearly related to cellular BrdUrd content than that achieved after acid DNA denaturation. However, cell loss is sufficiently severe with the thermal denaturation procedure that it may not be applicable to all cell types.

Animals↗
PubMed1985

Double-beam autocompensation for fluorescence polarization measurements in flow cytometry.

The degree of depolarization of fluorescent light emitted from an organic dye, which is used as molecular probe, is a powerful tool in probing the microenvironment. By fluorescence depolarization the macromolecular structure can be investigated as well as the the mobility of the marker molecule itself or of the complex formed by the probe. Additional information such as energy transfer rates, donor-acceptor distances, and orientations are also measurable. These data are of particular interest if they can be measured from whole cells. Using flow cytometry, we can analyze a large number of cells with high statistical significance in a short period of time. We describe a newly developed double-beam epi-illumination arrangement for fluorescence polarization measurements that uses an autocompensation technique. This new technique permits the various depolarizing effects within the optical as well as the electronic components of the system to be continually compensated for on a cell by cell basis. Simultaneous measurements of other cell parameters for cell cycle analysis by total fluorescence intensity remains possible. The sensitivity of the system to measure polarization was determined as +/- 0.006 p (0 less than or equal to p less than or equal to 0.5 in isotropic media), which amounts to +/- 1.2% of the maximum p value. Polarization data for latex microspheres plotted in the histogram mode were measured with a standard deviation of 0.006, which proved the high resolution and the high performance of the system.

Animals↗
PubMed1981

Analysis of urine sediments after kidney transplantation.

We applied multiparameter flow cytometry for the first time to the classification of cells and particles of urine sediments after kidney transplantation. After fluorescent staining with acridine orange, cells and particles were checked for their size and their peak intensity of green and red fluorescences, known to be related to the amounts of intracellular DNA and intracellular RNA, respectively. Urine sediments were tested daily during a period of up to three weeks. Occurrence and disappearance of certain classes of cells and particles were found to be similar among different patients during normal healing. The results from patients without rejection of the graft are suggested as baseline data, differences from which should be detectable with high sensitivity due to the large number of cells being examined.

Cytological Techniques↗
PubMed1981

Principles of fluorescence polarization measurements.

Fluorescence polarization measurements using high-speed single-cell flow cytometry have found increasing use in cellular biology. In most flow systems, the detection axis generally is aligned orthogonally to the direction of flow and the excitation axis, and asymmetric apertures along the excitation and emission axes by cylindrical lenses and/or nonplanar transition between optical indices complicate the numerical correction of systematic depolarization effects. In addition, recent studies on fluorescence emission of structured particles have shown remarkable anisotropies of polarized fluorescence emission dependent on the direction of the detection axis.

Chlorophyta↗
PubMed1980

Epi-illumination optical design for fluorescence polarization measurements in flow systems.

An epi-illumination design for fluorescence polarization measurements is introduced in flow cytometry with the optical axis orthogonally aligned to the cell stream. Various optical components and designs are discussed with respect to their influence on polarization measurements. Using the epi-configuration, paired measurements with the direction of polarization of the exciting light changed orthogonally are proposed for the compensation of system anisotropies and electronic mismatch. Large aperture corrections are employed for the excitation as well as for the emission pathway. Additional parameters such as fluorescence at 90 degrees, multiangle light scattering, and high precision cell-sizing by internally calibrated time of the flight measurements, as described previously, remain available with the design proposed here. Fluorescent latex microspheres, stained intracellular DNA, and algae have been used to test performance.

Cytological Techniques↗