bluuebunny/crossref_metadata_2025_split huggingface dataset:
Condensation of DNA in situ in metaphase chromosomes induced by intercalating ligands and its relationship to chromosome banding
Authors: Darzynkiewicz Zbigniew , Kapuscinski Jan 
Published: August 2005

AbstractInteractions of certain intercalating cationic ligands with nucleic acids result in the formation of products that undergo condensation and agglomeration; this transition in solution can be monitored by light‐scatter measurements. In the present study, using such intercalators as the antitumor drug mitoxantrone or fluorochromes acridine orange and quinacrine, we induced condensation of DNA in situ in Chinese hamster chromosomes. The in situ products scattered light and could be detected by darkfield‐ or phase‐contrast microscopy. In the darkfield the complexes had a characteristic granular appearance and often generated a banding pattern on the chromosomes. In contrast, condensation of DNA in situ by the nonintercalating polyvalent cations (Co3+, spermine4+), while enhancing the chromosome's image contrast, did not produce the granular products or the banding.The condensation of free DNA, single or double stranded, natural or synthetic, the later of various base composition and configuration, was also measured in solution. The condensation in solution and in situ was observed at similar concentrations of the respective ligands. The intercalating dye ethidium bromide, which did not condense DNA in solutions of moderate and high ionic strength, also did not generate the granular products or banding on chromosomes. The data also show that both base composition and configuration are important factors in determining the sensitivity of DNA to condensation by particular intercalating ligands.The studies suggest that the phenomenon of DNA condensation by intercalating dyes, which shows a high degree of specificity with respect to primary and secondary structures of DNA, may be associated with mechanisms of chromosome banding induced by the intercalating thiazine dyes in Giemsa staining or by quinacrine. Observation of chromosome banding based on light‐scatter detection in darkfield microscopy allows the study of interactions between DNA and the ligands that neither fluoresce nor generate colored products. This principle of chromosome “counter‐staining” can be explored by flow cytometry.
Light scattering and cell volumes in osmotically stressed and frozen‐thawed cells
Authors: McGann Locksley E. , Walterson Michele L. , Hogg Lori M. 
Published: August 2005

AbstractRecent reports, indicating that under some conditions the intensity of light scattering from cells is a nonlinear function of cell volume, have led to the widespread generalization that intensity of low‐angle light scattering indicates cell size. This study was performed to measure the relationships between light scattering and cell volumes in anisotonic solutions and after a freeze‐thaw stress.Cell volumes in isolated human lymphocytes, human granulocytes, and hamster fibroblasts were deliberately altered by exposure to anisotonic solutions. Boyle‐vant Hoff plots of cell volume as a function of inverse osmotic pressure showed that the cells behaved as osmometers. Similar plots of right‐angle and low‐angle light scattering showed that the intensity of light scattering varied inversely with cell volume.In other experiments where cells were frozen without cryoprotectant at various sub zero temperatures to −25°C and then thawed rapidly, cell viability decreased progressively with decreasing temperature, as did the intensity of both low‐angle and right‐angle light scattering, although cell volumes remained relatively constant.The intensity of both low‐ and high‐angle light scattering varied inversely with cell volumes in hypertonic and hypotonic solutions, but cell damage induced by freezing and thawing resulted in significant reductions in the intensity of low‐angle light scattering with little change in cell volume.These observations show that light scattering and cell volumes can vary independently, and they underline the need for a better understanding of the phenomenon of light scattering from living cells.
Nonselective enrichment for yeast adenine mutants by flow cytometry
Authors: Bruschi Carlo V. , Chuba Paul J. 
Published: August 2005

AbstractThe expression of certain adenine biosynthetic mutations in the yeast Saccharomyces cerevisiae results in a red colony color. This phenomenon has historically provided an ideal genetic marker for the study of mutation, recombination, and aneuploidy in lower eukaryotes by classical genetic analysis. In this paper, it is reported that cells carrying ade 1 and/or ade2 mutations exhibit primary fluorescence. Based on this observation, the nonselective enrichment of yeast cultures for viable adenine mutants by using the fluorescence‐activated cell sorter has been achieved. The advantages of this approach over conventional genetic analysis of mutation, recombination, and mitotic chromosomal stability include speed and accuracy in acquiring data for large numbers of clones. By using appropriate strains, the cell sorter has been used for the isolation of both forward mutations and chromosomal loss events in S. cerevisiae. The resolving power of this system and its noninvasiveness can easily be extended to more complex organisms, including mammalian cells, in which analogous metabolic mutants are available.
Detergent treatment of dictyostelium discoideum cells allows examination of internal cell type‐specific antigens by flow cytometry
Authors: Bernstein R. L. , Browne L. H. , Yu S. C. , Williams K. L. 
Published: August 2005

AbstractMonoclonal antibodies are used extensively in flow cytometry to identify subpopulations of cells differing in surface antigens. Conventional studies on living cells do not allow analysis of internal antigens, because antibody molecules do not pass through an intact plasma membrane. It is important for developmental studies on Dictyostelium discoideum that not only surface but also internal antigens be analysed. Here techniques are reported that make possible such studies by permeabilising cells with mild detergent treatments using digitonin. Flow cytometer profiles of unfixed cells show that antigens recognised by two monoclonal antibodies, MUD102 and MUD3, are found inside subpopulations of cells in the D. discoideum slug. Double‐labelling experiments were carried out to demonstrate that the antigens recognised by these antibodies are present inside prespore but not prestalk cells. The detergent treatment leads to loss of forward‐angle light scatter, but 90° light scatter of cells is not greatly affected. While fixed cells sometimes gave satisfactory results, internal labelling did not reliably demonstrate the two subpopulations observed with unfixed cells.
Fringe‐scan flow cytometry
Authors: Mullikin J. , Norgren R. , Lucas J. , Gray J. 
Published: August 2005

AbstractWe describe the development of a scanning flow cytometer capable of measuring the distribution of fluorescent dye along objects with a spatial resolution of 0.7 μm. The heart of this instrument, called a fringe‐scan flow cytometer, is an interference field (i.e., a series of intense planes of illumination) produced by the intersection of two laser beams. Fluorescence profiles (i.e., records showing the intensity of fluorescence measured at 20 ns intervals) are recorded during the passage of objects through the fringe field. The shape of the fringe field is determined by recording light scatter profiles as 0.25 μm diameter microspheres traverse the field. The distribution of the fluorescent dye along each object passing through the fringe field is estimated from the recorded fluorescence profile using Fourier deconvolution. We show that the distribution of fluorescent dye along microsphere doublets and along propidium iodide stained human chromosomes can be determined accurately using fringe‐scan flow cytometry. The accuracy of fringe‐scan shape analysis was determined by comparing fluorescence profiles estimated from fringe‐scan profiles for microspheres and chromosomes with fluorescence profiles for the same objects measured using slit‐scan flow cytometry.
Flow cytometric detection of herpes simplex virus type 2 infected and transformed cells by immunoenzymatic and by indirect immunofluorescence staining
Authors: Goolsby Charles , Gay Helen , Docherty John J. , Todd Paul 
Published: August 2005

AbstractThe glucose oxidase antiglucose oxidase (GAG) immunoenzymatic staining procedure has been used to detect herpes simplex virus (HSV) antigens microscopically. In this study, the GAG procedure was adapted to cells in suspension, and its potential usefulness in flow cytometry was examined. HSV‐2 infected monkey kidney and HSV‐2 transformed mouse cells were stained using antisera to HSV‐2 or to an HSV‐2 specific protein with a molecular weight of 38 Kd, respectively, with the GAG procedure. Flow cytometric analysis of the GAG stained cells was then performed by the measurement of scattered light intensity in the angular intervals l°−2°, 2.5°−19°, and 3°−6°. The greatest scattered light intensity decrement caused by staining occurred in the 3°−6° angular interval, as predicted by previous work. In infected cells, which stain intensely by immunofluorescence, the difference between positively and negatively stained cells was adequate for detecting infected cells using the GAG method; however, this was not the case for the lightly staining transformed cells. The indirect immunofluorescence method of analysis of the same populations was superior to the scattered light method of analysis of the GAG stained infected and transformed cells.
High‐speed photodamage cell selection using bromodeoxyuridine/Hoechst 33342 photosensitized cell killing
Authors: Herweijer Hans , And Willem Stokdijk , Visser Jan W. M. 
Published: August 2005

AbstractOne of the major drawbacks of droplet sorting in a flow cytometer is the relatively low sorting speed. Thus, we have developed an alternative, faster sorting technique: photodamage cell sorting. In a photodamage cell sorter all unwanted cells, as detected with the first, measuring laser, are killed with the second, damaging laser. Thus, the cells need to be photosensitive to the second laser. In addition, a mechanism is needed to switch this laser on and off based on the sorting criteria. In our photodamage cell sorter, the ZAPPER, we use an acousto‐optic crystal to switch the laser beam. Cells are made photosensitive by vital staining with photosensitizers. With cells grown in the presence of 5‐bromo‐2′‐deoxyuridine (BrdUrd) and stained with Hoechst 33342 (H42) at least a 5‐decade cell reduction is accomplished after irradiation with 400 mW UV light. With this system, sorting rates have been achieved of 30,000 cells per second. Due to the selection based on photodynamic killing, this sorting technique is restricted to the selection of viable cells.Photodamage cell sorting seems well suited for isolating viable cells occurring in low percentages or for the sorting of large numbers of cells. Another application can be the sorting of large or fragile cells.
Flow cytometric measurement of pollutant stresses on algal cells
Authors: Berglund Deborah L. , Eversman Sharon 
Published: August 2005

AbstractThe lichen Usnea fulvoreagens (Räs.) Räs. was treated with four pH levels (5.5, 4.5, 3.5, and 2.5) of simulated acid rain (sulfuric acid, nitric acid, and a 1:1 combination of both) and automobile exhaust. The samples were dissociated and analyzed by a Becton‐Dickin‐son FACS 440 flow cytometer. Analyses included measurement of chlorophyll autofluorescence and fluorescence due to uptake of fluorescein diacetate (FDA) and calcofluor white M2R (CFW). Cell parameters measured were esterase activity (FDA), membrane permeability (FDA, CFW), and intracellular pH (FDA). Mean fluorescence intensity from FDA staining and numbers of events were incorporated with autofluorescence information to produce a “stress index” of relative cell stress. Results indicated that highly stressed samples (lower pH treatments and greater exposure to exhaust) exhibited a low “stress index” of FDA fluorescence.
Cyclosporin a does not inhibit the PHA‐stimulated increase in intracellular Ca2+ concentration but inhibits the increase in e‐rosette receptor (CD2) expression and appearance of interleukin‐2 receptors (CD25)
Authors: Redelman Doug 
Published: August 2005

AbstractThe immunosuppressive drug cyclosporin A (CsA) inhibits mixed lymphocyte responses, blocks the generation of cytotoxic T lymphocytes, and inhibits the T lymphocyte proliferative response stimulated by polyclonal activators such as phytohemagglutinin (PHA). Nevertheless, there have been contradictory reports attempting to explain the mechanism(s) for this immunosuppressive activity. In the current studies, human peripheral blood mononuclear cells (PBM) were stimulated with PHA in the presence or absence of CsA. Flow cytometric examination of PBM loaded with the Ca2+‐sensitive dye Indo‐1 showed that concentrations of CsA sufficient to inhibit 90‐100% of tritiated thymidine incorporation had no effect on the PHA‐stimulated increase in the intracellular Ca2+ concentration ([Ca2+]i). Likewise, inhibitory amounts of CsA had virtually no effect on the increase in cell volume that occurs during T lymphocyte activation. These results were not altered by pretreating the PBM with CsA for 30 min at 37°C prior to adding the PHA. On the other hand, inhibitory concentrations of CsA prevented the expression of receptors for T cell growth factor (interleukin‐2, IL‐2), as measured by monoclonal antibodies to CD25 after 16‐24 hr incubation. In like manner, CsA also prevented the increase in the expression of the E‐rosette receptor (CD2) on these same cells. If cultures containing PHA and inhibitory amounts of CsA were incubated for 40‐72 h, there was partial recovery both of proliferative activity and of the expression of CD25 and CD2. Thus, CsA does not appear to affect the initial activation signal(s), but does interfere with one or more subsequent events necessary to initiate the appearance of “activation antigens.”
Cell cycle distribution in‐vivo of mononuclear blood cells of renal allograft recipients
Authors: Walle Alexander J. , Wong George Y. 
Published: August 2005

AbstractIn 47 healthy blood donors (controls) and 29 renal allograft recipients (patients) the relative contents of RNA and DNA of peripheral blood mononuclear (PBM) cell populations were estimated from the intensities of green and red fluorescences emitted by complexes that form with DNA and RNA, respectively, after staining the cells with the metachromatic dye, acridine orange. Based on the correlated DNA and RNA estimates for large numbers of cells, the percentages and the relative RNA contents of cells in particular compartments of the cell cycle were determined. PBM cell populations of controls contained < 0.5% proliferating (SG2M) cells with highly variable relative RNA contents. Among controls, neither percentages nor relative RNA contents of SG2M‐cells were correlated with percentages or relative RNA contents of G1‐cells with an RNA content 2 (2SD) or 3 (3SD) standard deviations above the mean of the entire GO/1‐cell population. Unlike controls, PBM cell populations of patients contained significantly higher percentages of SG2M‐cells which were significantly correlated with the relative coefficients of variation (rCV) of the F530 histograms of GO/1‐cells; the rCV represents the ratio of a patient's CV to a control's CV. Moreover, significant statistical correlations existed between percentages and relative RNA contents of 2SD‐, 3SD‐, SG2M‐and GO/1‐cells, suggesting a well‐orchestrated progression of cells through the cell cycle. Different pairs of correlated parameters characterized clinically stable, acutely rejecting, and infected patients. Finally, the data of this pilot project suggest that two features of GO/1‐cells, namely the rCV and the mean relative RNA content may carry the information necessary to predict the cell cycle distribution of PBM cells of stable, rejecting, or infected renal allograft recipients.
Altered nucleic acid contents of mononuclear cells in blood of renal allograft recipients
Authors: Walle A. J. , Wong G. Y. , Suthanthiran M. , Rubin A. L. , Stenzel K. H. , Darzynkiewicz Z. 
Published: August 2005

AbstractThe DNA and RNA contents of blood mononuclear cell populations of 29 cadaver renal allograft recipients and 49 blood donors (controls) were estimated by acridine orange flow cytometry (AO FCM) to assess their cell cycle status. All patients received azathioprine and prednisone for immunosuppression. The patients represented three clinical categories: clinically stable patients, those with acute rejections (clinically overt or impending), and those with infections. Three cell cycle compartments were analyzed for percentage (%) and RNA content (R) of cells: GO/1, consisting of all cells with diploid DNA content; 2 S.D., consisting of cells with diploid DNA content and RNA content 2 standard deviations above the mean RNA content of controls; and SG2M, consisting of cells with a DNA content higher than that of GO/1 cells. The relative coefficient of variation (rCV) of the DNA distribution of GO/1 cells was also determined. In such cell cycle evaluations, the means of rCV and SG2M% of stable recipients were significantly different from those of blood donors. Multivariate analysis of the variables of the three categories of patients resulted in the tentative formulation of two simple logistic equations: one that differentiates stable patients from those with impending or overt rejections based on 2SD% and another one that distinguishes infected patients from those with impending or overt rejections based on SG2M% and RG0/1.
Image analysis applied to proliferating cells in malignant lymphoma
Authors: Souchier C. , Ffrench M. , Scoazec J. Y. , Bryon P. A. , Berger F. 
Published: August 2005

AbstractThis study describes a method for obtaining the distribution of a given cell type in histological sections, as revealed by a specific immunological marker. Two hundred fifty‐six contiguous microscopic fields were analysed, and a cellular map was automatically constructed. The resulting images were stored on magnetic media for subsequent quantification and statistical tests. The method was applied to the analysis of 20 small cleaved‐cell lymphomas in which the monoclonal antibody Ki‐67 was used to mark proliferating cells. Clusters of proliferative cells were observed in both diffuse and nodular lymphomas. Cellularity and the percentage of marked cells were not significantly different in the two types of tumors.
Fixation and long‐term storage of human lymphocytes for surface marker analysis by flow cytometry
Authors: Lal Renu B. , Edison Linette J. , Chused Thomas M. 
Published: August 2005

AbstractA method to preserve stained human lymphocytes for subsequent cell surface analysis by flow cytometry (FCM) is described. Cells stained with fluorescein isothiocyanate (FITC) and phycoerythrin (PE)‐conjugated monoclonal antibodies and then fixed in 1% paraformaldehyde, followed by extensive washing and resuspension in 1% BSA medium, could be stored at 4°C for at least 2 weeks prior to FCM analysis without significant alteration in the light scatter or fluorescence properties of the cells. Furthermore, the method was also suitable for analying lymphocytes that express T‐cell activation markers in certain disease conditions. In addition, we have identified monoclonal antibody combinations that discriminate different lymphocyte subsets that are satisfactory for multiparameter analysis after 2 weeks of storage. This method should prove useful for enumerating lymphocyte subsets in field study sites remote from flow cytometry laboratories.
DNA flow cytometry of endoscopically examined colorectal adenomas and adenocarcinomas
Authors: Giaretti Walter , Bruno Silvia , Geido Elio , Vinci Angela Di , Sciallero Stefania , Aste Hugo 
Published: August 2005

AbstractDNA ploidy of 64 colorectal adenomas and 49 adenocarcinomas, examined endoscopically, was studied by flow cytometry. We found DNA aneuploidy in none of the 105 normal mucosa samples (0%), in 20 adenomas (31%), and in 36 adenocarcinomas (74%). DNA ploidy of adenomas correlated with size (P = 0.02) and degree of dysplasia (P < 0.01) but not with histologic type. Adenomas had a 45% incidence of DNA aneuploid stem lines in the DNA index range of 0.80–1.20, compared with 8% in the case of adenocarcinomas. The distribution of the DNA index values of adenocarcinomas was approximately normal, with a mean value 1.63 × 0.28. The mean DNA index for the three cases of “carcinoma in adenoma” with invasion of the stalk of the adenoma was 1.52 × 0.18. These results, using DNA flow cytometry, provide evidence for the progression of colorectal adenoma to adenocarcinoma. The classification of adenomas according to DNA ploidy may be information of considerable practical value to the clinician in predicting risk of further adenomas and/or risk of cancer.
Specific nuclear elimination in polyploid plasmodia of the slime mold physarum polycephalum
Authors: Werenskiold A. K. , Schreckenbach Th. , Valet G. 
Published: August 2005

AbstractIn growing plasmodia of the myxomycete Physarum polycephalum (G2‐phase), three distinct classes of nuclei with a relative DNA content of 1x, 2x, and 4x are observed in the presumed haploid strain CL. The 2x and 4x species comprise up to 35% and 5% of the nuclei. Quantitative cytofluorometric studies of nuclei isolated in either G2‐ or S‐phase or after FUDR treatment (G1 arrest) show that the three nuclear populations undergo a synchronous mitotic cycle and that the relative DNA content of the nuclear fractions in G‐2 phase reflects the 2c, 4c, and 8c state.The heterogeneity of the nuclear population does, however, seem to be restricted to the growth phase. During a starvation period of 4 days that always preceeds sporulation (and also meiosis), the 4c nuclear population is reduced to 7%, 8c nuclei are no longer detected. These results suggest that a mechanism exists in Physarum for the selective detection and elimination of polyploid nuclei.
Improved resolution of flow cytometric measurements of hoechst‐ and chromomycin‐A3‐stained human chromosomes after addition of citrate and sulfite
Authors: Engh Ger Van Den , Trask Barb , Gray Joe , Lansdorp Peter 
Published: August 2005

AbstractThe resolution of bivariate flow karyotypes of human chromosomes stained with Hoechst 33258 and chromomycin A3 can be increased by adding sodium citrate and sodium sulfite to the chromosomes shortly before measurement. A flow karyotype of a patient with chronic myelocytic leukemia is shown to illustrate that the addition of these compounds allows high‐resolution measurements to be made and evaluated reliably from clinical samples.