bluuebunny/crossref_metadata_2025_split huggingface dataset:
Improved flow cytometry of cellular DNA and RNA by on‐line reagent addition
Authors: Pennings Arie , Speth Paul , Wessels Hans , Haanen Clemens 
Published: August 2005

AbstractAcridine orange (AO) staining enables flow cytometric measurement of cellular DNA and RNA. With the conventional procedure, red and green fluorescence intensities alter considerably in time, affecting the reliability and reproducibility of the results. A standardized simple technique of AO staining is described that keeps the time between addition of detergent and AO and the subsequent measurement constant. Measurements by means of this on‐line method appear to be much more reproducible in comparison to those obtained using the conventional procedure.
Multiple wavelength illumination in flow cytometry using a single arc lamp and a dispersing element
Authors: de Grooth B. G. , van Dam M. , Swart N. C. , Willemsen A. , Greve J. 
Published: August 2005

AbstractThe principle of a multiple wavelength illumination method for flow cytometers, based upon a combination of a helium‐neon laser and an arc lamp as illumination sources is described. By using a prism, the light from the arc lamp is dispersed and the different colors are imaged at different places on the sample stream. The small angle light scattering from the helium‐neon laser light is measured as a relevant parameter and serves as a trigger signal for subsequent measurements of fluorescence or scattering of light from the arc lamp. Two experimental systems are described utilizing this principle: a system where the emission is detected orthogonally with respect to the direction of the illumination beams, and an epi‐illumination system.With the orthogonal set‐up multiple wavelength right angle scattering measurements are possible. This is illustrated by showing that the orthogonal scattering from erythrocytes is strongly dependent on the illumination wavelength. It is further shown that the apparatus is suitable for the measurement of intracellular pH using the pH dependence of the excitation spectrum of fluorescein.The epi‐illumination system allows excitation of two (or more) fluorescent dyes with different excitation spectra. In this case the emission spectra of the fluorescent dyes may overlap substantially. This is shown by simultaneous measurement of DNA and protein of Chinese hamster lung cells using mitramycin and tetramethyl rhodamin isothiocyanate (TRITC).
Evaluation of DNA and nuclear protein features for their use in describing normal and malignant endometrium
Authors: Oud Peter S. , Katzko Michael W. , Pahlplatz Martin M. M. , Vooijs G. Peter 
Published: August 2005

AbstractDifferent feature sets (geometric, densitometric, and textural) derived from DNA and nuclear protein staining were evaluated for their use in describing atrophic, secretory, and proliferative endometrium, and well‐differentiated stage I and moderately differentiated stage I adenocarcinomas of the endometrium. It was found that the pattern of significant differences among these groups varied between feature sets, while remaining consistent within a set of features. The DNA density and run‐length features were not very effective in describing group mean differences, whereas the co‐occurrence features revealed significant differences among most groups. The protein run‐length features were the only ones that consistently showed a difference between proliferative endometrium and well‐differentiated adenocarcinomas. Analyses repeated on only cells in the G0/G1 DNA region improved the differentiation between moderately differentiated adenocarcinomas and the other groups. It was concluded that the use of DNA and nuclear protein texture features are effective in describing group differences that cannot be described by DNA content only.
Visualization of DNA loops in nucleoids from HeLa cells: Assays for DNA damage and repair
Authors: Roti J. L. Roti , Wright W. D. 
Published: August 2005

AbstractAn assay for visualization of DNA loops undergoing supercoiling changes has been developed. The assay utilizes the fluorescent dye, propidium iodide (PI), which intercalates into the DNA and under the proper conditions causes the supercoiling status of the DNA to change. Thus, the DNA can be seen as a fluorescent halo that changes diameter with PI concentration. At low PI concentrations (0–7.5 μg/ml) the supercoils are relaxed with increasing PI, while at higher PI concentrations (7.50–50 μg/ml) supercoils in the opposite winding sense are rewound with increasing PI. When HeLa cells were irradiated with 1–20 Gy of 137Cs γ‐rays, the ability to rewind the DNA supercoils was inhibited in a dose‐dependent manner, presumably because of the presence of radiation‐induced DNA strand breakage, which removed the topological constraints on the DNA loops. These lesions were repaired rapidly during post‐irradiation incubation. The ability of the DNA loops to be rewound was restored within 8 min after 10 Gy of γ‐irradiation, such that no difference from control cells could be detected. The halftime for repair of the radiation‐induced lesions that inhibit DNA rewinding was similar to that for repair of DNA single strand breaks. The assay has certain advantages over current methods for assaying DNA damage in that it involves measurement of single cells and it does not require the DNA to be labeled with radioactive precursors.
Quantification of the nonspecific intercellular transfer of fluorescent molecules between labeled and unlabeled rat thymocytes
Authors: Capo Christian , Mege Jean Louis , Benoliel Anne Marie , Mishal Zohair , Bongrand Pierre 
Published: August 2005

AbstractFluorescein isothiocyanate has been used to label normal or tumor cells in order to study their migration in vivo. This requires that any transfer of fluorescence to neighbouring cells be carefully ruled out. The aim of the present report is to demonstrate the possibility of a transfer of fluorescein or fluorescein‐bound molecules between untreated and labeled cells. When normal rat thymocytes were co‐incubated with labeled cells (about 5 × 106 fluorescein molecules/cell) under continuous agitation or exposed to supernatants of these labeled cells, they bound an average of 104 fluorescein molecules. When the incubation was done on cell pellets after centrifugation, this transfer was increased tenfold. Hence, intercellular molecule exchange may occur in the absence of any specific interaction.
Flow cytometric analysis of DNA in cells obtained from deparaffinized formalin‐fixed lymphoid tissues
Authors: McIntire Timothy L. , Goldey Stacia H. , Benson Neal A. , Braylan Raul C. 
Published: August 2005

AbstractFlow cytometric analysis of DNA content was performed on nuclear suspensions prepared from fresh and from paraffin‐embeded, formalin‐fixed lymphoid tissues. We confirmed previous reports that it is possible to obtain nuclear suspensions from deparaffinized, formalin‐fixed tissues, suitable for DNA analysis by flow cytometry. We observed a tendency for a larger coefficient of variation (CV) of the DNA measurements in the fixed tissues than in the unfixed material causing abnormalities in 2 of 19 lymphomas to become undetectable. Furthermore, samples from different paraffin blocks of a single tumor with an extra G1 (hyperdiploid) peak showed marked differences in the CV of the hyperdiploid peak while the CV of the diploid peak was similar in all samples. In both benign and malignant lymphoid tissues, the S‐phase fraction was higher in paraffin‐embedded tissues than in unfixed cells. This difference could be attributed to 4′, 6′‐diamidino‐2 phenylindole dihydrochloride (DAPI), a DNA‐binding dye commonly used in this technique. Nevertheless, intermediate and high grade lymphomas from paraffin‐embedded tissues generally showed a greater S‐fraction than low grade lymphomas, a similar observation as with unfixed tissues. Therefore, DNA content analysis of nuclei extracted from paraffin sections may be inadequate to resolve slight aneuploidy, but the measurement of S‐fraction size may remain diagnostically or prognostically valuable. Large retrospective studies will be necessary to determine the clinical impact of this technique in the analysis of lymphomas.
Surface glycoproteins of differentiating neuroblastoma cells analyzed by lectin binding and flow cytometry
Authors: Notter Mary F. D. , Leary James F. 
Published: August 2005

AbstractCell surface glycoprotiens of mitotic neurobalstoma cells and cells differentiated by prostaglandin cyclic adenosine monophosphate treatment were quantified by flow cytometric analysis and specific fluorescent lectins. No differences in fluorescent lectin binding were seen between suspensions of mitotically active and differentiated N2AB‐1 cells following exposure to either fluorescein (FL)‐labeled soy bean agglutinin (FL‐SBA) specific for N acetyl galactosamine or FL‐concanavalin A (FL‐CON A) which binds to mannose residues. These lectins, however, were shown to bind specifically to these cells as revealed by competitive blocking studies with hapten sugars. When FL Ulex europaeus (FL‐UEA) specific for fucose was reacted with control or differentiated cells, no binding was seen even with an increased dose of lectin before or after enzyme treatment. However, differentiated N2AB‐1 cells, reacted with FL‐wheat germ agglutinin (FL‐WGA) specific for N acetyl glucosamine, bound more FL‐WGA than that seen for control cultures. Furthermore, specific sites for FL‐WGA were shown to be saturable and were lost upon pretreatment of cells with neuraminidase. Neuraminidase pretreatment revealed masked sites for FL‐CON A and FL‐SBA since binding was increased at least twofold for these lectins on mitotic and differentiated cells. These data indicate that single cell measurements of surface glycoproteins can be made on living neural cells and that differentiation induces an increase in cell surface N‐acetyl glucosamine residues.
A rapid and simple estimation of cell cycle parameters by continuous labeling with bromodeoxyuridine
Authors: Sasaki Kohsuke , Murakami Tomoyuki , Takahashi Manabu 
Published: August 2005

AbstractThe DNA synthesis time (Ts) and other related cell cycle parameters were roughly estimated in HeLa cells labeled with bromodeoxyuridine (BrdUrd) for various durations by using the flow cytometrical technique. The labeling indices increased in proportion to time after addition of BrdUrd. The Ts can be calculated from the slope of the regression line obtained by plotting the serial labeling indices against the labeling time and was equivalent to the value determined by fraction labeled cells in mid S‐phase (FLSm) method. These parameters would be determined by only two samples labeled for different times. This simple method using BrdUrd provides rough but rapid estimation of Ts and other cell cycle parameters without complicated mathematical procedures, in addition to cell cycle partition of cell populations.
Technique and staining optimization leucoconcentration
Authors: Pierrez J. , Guerci A. , Guerci O. 
Published: August 2005

AbstractIn cytometric clinical application, it is important to obtain cell suspensions rapidly with as little cytological alteration as possible. A procedure has been achieved to prepare cell suspensions for flow cytometric analysis. The leucoconcentration technique, first described by Herbeuval for cytologic analysis, has been modified to be applied in cytometry. This technique involves Saponin lysis of red cells of peripheral blood or bone marrow samples that have been previously fixed with picric acid alcohol solution. Cells in suspension are not shifted and tinctorial affinity is not modified.Then cells have been stained with Mithramycin. Each parameter defined by Crissman has been analyzed to define the best staining conditions.The availability of Leucoconcentration with Mithramycin‐DNA‐staining permits determination of cell cycle with a fine resolution.
Thiazole orange: A new dye for Plasmodium species analysis
Authors: Makler M. T. , Lee L. G. , Recktenwald D. 
Published: August 2005

AbstractA rapid sensitive method for the determination of Plasmodium falciparum in in vitro culture is presented. The technique employs a fluorescent flow cytometer equipped with a 15‐mwatt argon laser that emits light at 488 nm and a membrane‐permeable fluorochrome thiazole orange (TO) that stains RNA. Parasitized red cells are stained by suspending them in 1 ml of phosphate‐buffered saline (PBS) containing 10×5 M of TO and incubating this mixture for 15 min in the dark at room temperature. The stained cells may be analyzed fresh or after fixation with 1% paraformaldehyde/PBS or 0.25% glutaraldehyde/PBS. Alternatively the cells may be fixed first and then stained. There is excellent correspondence between the number of fluorescent‐labeled parasitized red cells and Giemsa‐stained cells.
Characterization of a carbocyanine derivative as a fluorescent penetration probe
Authors: Olive P. L. , Durand R. E. 
Published: August 2005

AbstractThe fluorescent carbocyanine dye 3,3‐diheptyloxycarbocyanine [DiOC7(3)], originally described as a membrane potential probe, penetrates poorly into multicell spheroids. Since the dye is retained in the cells following spheroid disaggregation, cells can be selected from different depths within the spheroid using fluorescence‐activated cell sorting. Characterization of the binding kinetics, stability, and toxicity of this probe were undertaken, and intercompared with Hoechst 33342. The optimum drug dose for achieving good separation of internal and external cells of spheroids is about tenfold lower than for Hoechst 33342, and like Hoechst, DiOC7(3) is toxic at concentrations at least tenfold higher than those required to produce a good gradient for cell separation. When cells are removed from the stain, cellular fluorescence decreases to half the initial intensity within 2 hours; however, unlike Hoechst, the carbocyanine dye does not transfer between cells.
Feulgen DNA stainability of bone tumors after demineralization
Authors: Bauer Henrik C. F. , Kreicbergs Andris 
Published: August 2005

AbstractMicrospectrophotometric DNA analysis of archival bone tumor tissue is often impeded by previous acid demineralization, which destroys Feulgen DNA stainability. To find an alternative to acid for prospective DNA studies of bone tumors in tissue sections, Feulgen stainability of fresh osteosarcoma specimens after demineralization in neutral EDTA was investigated. The reliability of DNA analysis of weakly Feulgen‐stained sections from archival tissue was also studied.Demineralization of four fresh specimens in EDTA slightly reduced Feulgen DNA stainability compared to nondemineralized preparations but did not affect the determination of ploidy level. Hydrolysis tests of one diploid and one hyperploid osteosarcoma showed that the staining relationship between control and tumor cells was not altered by EDTA pretreatment. For DNA studies of bone tumors requiring demineralization, EDTA offers a means of retaining nuclear Feulgen stainability.In 22 archival osteosarcoma specimens of varying Feulgen stainability, three different upper limits of light transmission (75, 85, and 95%) were applied to test the significance of background disturbances in relation to nuclear stain intensity. The relationship between the median total extinction of the control and tumor cell populations was not significantly affected by altering the upper transmission limit except in four poorly stained lesions. The control cells of these four specimens exhibited a median total extinction less than one‐third of the maximum encountered. The results suggest that weakly stained archival specimens can be tested for selecting those appropriate for ploidy determination.
Flow cytometric analysis of isolated rat liver nuclei during growth
Authors: Papa Stefano , Capitani Silvano , Matteucci Alessandro , Vitale Marco , Santi Patrizia , Martelli Alberto M. , Maraldi Nadir M. , Manzoli Francesco A. 
Published: August 2005

AbstractThe development of hepatocyte polyploidy in rats aged up to 4 months was analyzed by flow cytometry using both scatter and fluorescent parameters to distinguish DNA diploid and DNA tetraploid populations and to discriminate between parenchymal and non‐parenchymal compartments. The precise origin of each class of nuclei was assessed in whole liver homogenate using purified hepatocytes, obtained by liver perfusion followed by separation on Percoll gradient, and identifying the peaks corresponding to parenchymal nuclei. The results indicate that preparative procedures involving homogenization of the rat liver tissue caused loss of the DNA octaploid population. Data on the relative proportion of the different DNA ploidy elements during rat liver development, which are in good agreement with those observed by cell analysis by means of microspectrophotometry, indicate the usefulness of flow cytometry as a choice method for the analysis of ploidy distribution.
DNA synthesis rate and unlabeled cells with S‐phase DNA content in P388 leukemic cells in vivo studied by flow cytometry and cell sorting
Authors: Clausen O. P. F. 
Published: August 2005

AbstractDNA synthesis kinetics of P388 leukemic cells growing in ascites form in BDF1 hybrid mice were investigated during the periods of exponential growth and growth restriction. Incorporation of tritiated thymidine, and in some instances tritiated uridine, was studied by autoradiography in cells sorted from S‐phase fractions during DNA flow cytometry.During exponential growth continuous labeling with tritiated thymidine indicated a growth fraction of unity, whereas the growth fraction was about 30% during growth restriction. At this growth phase the majority of cells with S phase DNA content remained unlabeled after pulse labeling with tritiated thymidine or uridine, indicating that both the “salvage” and the “de novo” DNA synthesis pathways were blocked in most S‐phase cells.After pulse labeling with tritiated thymidine the DNA synthesis rate pattern was investigated by sorting of consecutive fractions of cells throughout the S phase followed by quantitative autoradiography. With exception of a reduced rate in the middle of S phase, the DNA synthesis rate increased as the cells progressed through S phase during exponential growth. In contrast, the DNA synthesis rate pattern had a relative peak in the middle of S phase during growth restriction, which is otherwise characterized by a low mean DNA synthesis rate.
Bivariate flow karyotyping of acute myelocytic leukemia in the BNML rat model
Authors: Arkesteijn G. J. A. , Martens A. C. M. , Jonker R. R. , Hagemeijer A. , Hagenbeek A. 
Published: August 2005

AbstractUnivariate as well as bivariate flow karyotyping has been performed on chromosome suspensions obtained from the Brown Norway myelocytic leukemia (BNML), a rat model for human acute myelocytic leukemia (AML). Flow karyograms were obtained from both the in vivo transplantable parent line and from an in vitro established cell line. Density gradient centrifugation performed on cells arrested in mitosis resulted in an enrichment of mitotic cells. Furthermore, with this procedure leukemic and nonleukemic cells could be separated. Univariate analysis with propididum iodide (PI) as a DNA stain revealed the position of the several tumor‐specific marker chromosomes in the in vitro cell line. Estimations of the peak position of the various chromosomes was done by comparing the univariate flow karyogram with a computer‐simulated karyogram from the BNML that was derived from the mean length of the individual chromosomes in conventionally prepared metaphase slides. By comparing the bivariate flow karyogram of the in vivo BNML cells with the flow karyogram of normal BN cells, it was clearly demonstrated which peaks are involved in the altered chromosomal pattern of the BNML. No differences were found between the flow karyograms of the in vitro‐and the ex vivo‐derived chromosome suspensions in this rat leukemia model.
Quantitative description of nuclear morphology in assessing resistance of sarcoma 180 to adriamycin
Authors: Komitowski D. , Sonka J. , Schmitt B. , Muto S. 
Published: August 2005

AbstractResistance to adriamycin generally is explained through changes of cell/drug interactions that possibly reflect structural alterations of intracellular targets. One of the main targets of adriamycin is believed to be nuclear chromatin. In order to recognize chromatin alterations, we studied cell nuclei morphology and chromatin structure by means of digital image analysis. The studies were performed in both adriamycin‐sensitive and ‐resistant Sarcoma 180 cell lines which were cultured under growth‐stimulated and nonstimulated conditions. Using specially developed methods, we extracted parameters characterizing geometrical, optical, and structural properties of the cell nuclei from light microscopical images. The latter parameters concerned microscopical appearances of condensed chromatin and were described by features of high‐optical‐density regions. The results demonstrated that the quantitative criteria applied enabled the discrimination of sensitive and resistant cells. The most important parameters are the nuclear size, number, distribution, and optical density of condensed chromatin regions. In addition, the criteria permit recognition of changes related to differences in the growth conditions of the cells. The data of the image analysis suggest that adriamycin resistance in Sarcoma 180 cells is associated with characteristic patterns of cell nuclear morphology which can be described with a sufficient number of appropriate parameters. The advantages of image analysis are evident when these results are compared with the flow cytometric findings. The conclusion is that structural features of nuclear chromatin provide information essential for the assessment of drug resistance.
Effect of drug efflux blockers on vital staining of cellular DNA with hoechst 33342
Authors: Krishan Awtar 
Published: August 2005

AbstractThe present study shows that staining of certain live cells, e.g., adriamycin‐resistant P388 cells, by Hoechst 33342 is difficult because of the presence of a rapid efflux pump, which reduces intracellular dye concentration. Coincubation of these refractory cells in the presence of efflux blockers such as phenothiazines (trifluoperazine) or Ca++ channel blockers (verapamil) enhances dye retention and thus leads to generation of normal DNA distribution histograms. Laser flow cytometric data is confirmed by fluorometric assays, which show that P388/R cells retain one‐third the amount of Hoechst 33342, and coincubation with efflux blockers increases Hoechst retention to values similar to those of drug‐sensitive P388 cells. DNA histograms of mouse splenocytes incubated with Hoechst 33342 alone have a bimodal distribution possibly because of the presence of subpopulations that do not retain the fluorochrome owing to rapid efflux. Coincubation with an efflux blocker results in the generation of unimodal DNA histograms from these cells.These preliminary studies suggest that reduced retention of Hoechst 33342 in certain cell types (because of rapid efflux) can be blocked by efflux blockers, thus leading to generation of typical DNA distribution histograms.
Time, a quality‐control parameter in flow cytometry
Authors: Watson James V. 
Published: August 2005

AbstractTime has been used as a quality‐control parameter in our flow cytometer. This parameter was automatically incorporated into the list‐mode data base by hooking the computer clock directly into the acquisition logic. Quality control can then be checked by “replaying” fluorescence or light scatter data vs. time and any instrumental drift can be observed and degraded data can be excised. The technique is illustrated with a number of DNA data sets from a tissue culture cell line in which artificial perturbations were introduced during data acquisition to simulate potential causes of instrumental drift.