bluuebunny/crossref_metadata_2025_split huggingface dataset:
Denaturation, renaturation, and loss of DNA during in situ hybridization procedures
Authors: Raap A. K. , Marijnen J. G. J. , Vrolijk J. , van der Ploeg M. 
Published: August 2005

AbstractWith the aim of optimizing in situ hybridization methods, alkaline, acid, and thermal denaturation procedures have been studied for their ability to separate the DNA strands of nuclear DNA and for the DNA losses they induce. Isolated methanol/acetic acid‐fixed mouse liver nuclei have been used as a biological object. The results, obtained with acridine orange staining and microfluorometry, show that all denaturations studied lead to almost complete strand separation. Quantitative DNA staining and cytometry indicated that with heat and alkaline denaturation about 40% of the DNA is lost. Acid denaturation led to about 20% DNA loss. For the alkaline denaturation, the DNA retention could be improved to a 20% DNA loss by adding 70% ethanol to the denaturation medium. During hybridization, another 20% DNA loss occurs.When denatured nuclei are brought under annealing conditions, a rapid renaturation of a considerable fraction of the remaining DNA occurs. The extent of renaturation was dependent on the type of denaturation used. For the ethanolic alkaline denaturation, it was estimated to be 35%. Quantitative nonautoradiographic in situ hybridization experiments with acetylaminofluorene‐modified mouse satellite DNA showed that alkaline denaturation procedures are superior to the heat and acid denaturation.As proven by acridine orange fluorescence measurements, hybridization conditions can be designed that permit DNA. RNA hybridization under in situ DNA. DNA denaturing conditions. These conditions should be very useful, especially for in situ hybridization with single‐stranded RNA probes.
Chemosensitivity of human small cell carcinoma of the lung detected by flow cytometric DNA analysis of drug‐induced cell cycle perturbations in vitro
Authors: Engelholm Svend A. , Spang‐Thomsen Mogens , Vindeløv Lars L. , Brünner Niles A. 
Published: August 2005

AbstractA method based on detection of drug‐induced cell cycle perturbation by flow cytometric DNA analysis has previously been described in Ehrlich ascites tumors as a way to estimate chemosensitivity. The method is extended to test human small‐cell carcinoma of the lung. Three tumors with different sensitivities to melphalan in nude mice were used. Tumors were disaggregated by a combined mechanical and enzymatic method and thereafter have incubated with different doses of melphalan. After incubation the cells were plated in vitro on agar, and drug induced cell cycle changes were monitored by flow cytometric DNA analysis. Melphalan produced a dose‐related S phase accumulation in the two sensitive tumors, whereas no changes in the cell cycle distribution were found in the resistant tumor. The size of S phase accumulation correlated to the chemosensitivity in vivo. For low concentrations of melphalan, the S phase accumulation was accompanied by G2 + M accumulation. The results indicate that the method may be extented to sensitivity testing of human solid tumors, including screening for new agents.
Graphical presentation of multidimensional flow histogram using hexagonal segmentation
Authors: Kosugi Yukio , Ikebe Jun , Shitara Nobuyuki , Takakura Kintomo 
Published: August 2005

AbstractImprovements in the method of presenting a multidimensional histogram for flow cytometric data are described. Two‐dimensional data were systematically quantized into hexagonal subareas, which facilitated an accurate presentation of the distribution with a smaller number of bins and sample cells.
Cell‐cooling in flow cytometry by peltier elements
Authors: Göttlinger Christopher , Meyer Klaus L. , Weichel Walter , Müller Werner , Raftery Brian , Radbruch Andreas 
Published: August 2005

AbstractWe have built a cooling device for cell suspensions in flow cytometry that makes use of the Peltier effect (Barnard RD, Thermo electricity in Metals and Alloys, Taylor and Francis, London; Siemens‐Z 34:383–88, 1963). The prototype described here is used for cooling collection tubes during long‐duration cell sorting and is capable of maintaining a temperature of 2–5°C in a cell suspension of up to 3 ml. In general, Peltier element‐based cooling is useful for equilibrating the temperature of small volumes of fluids. Furthermore, Peltier element‐based cooling devices are easy to build and handle.
A comparison between flow cytometric ploidy investigation and chromosome analysis of 32 human colorectal tumors
Authors: Petersen Steffen E. , Friedrich Ursula 
Published: August 2005

AbstractThe correlation between flow cytometric ploidy investigation and classic chromosome analysis was studied in 32 human colorectal tumors. Flow cytometry was performed by nuclei isolation and DNA staining with ethidium bromide. Chromosome analysis was done after incubation with colcemid. In 12 cases, chromosome identification was possible by grouping according to the Denver system or by Q‐banding. Generally, the measured DNA content corresponded well with the content expected from chromosome analysis, giving an average difference of 4%. In nine tumors, the measured DNA content was 4‐18% higher than expected. Some of these discrepancies could be due to difficulties in identifying the corresponding cell populations in heterogeneous tumors. However, in general the number of cell populations and their quantitative representation by the two methods were statistically well correlated.The results indicate that flow cytometric ploidy investigation of colorectal tumors with the present technique is a reliable method, but also that a combination of both techniques may yield additional information about tumor cytogenetics.
Growth and analysis of vascular smooth muscle on microspheres
Authors: Dennis Patricia A. , Wolley Robert , Taylor Nancy S. , Moyer Carolyn F. 
Published: August 2005

AbstractProtocols for culturing vascular smooth muscle cells (VSMCs) on polystyrene microspheres, assessing VSMC contractility while adhered to the microspheres, and analyzing VSMCs cultured on microspheres by flow cytometry are described. These procedures preserve optimal VSMC viability and function, while providing a method for assessing these cells within the parameters required for analysis by flow cytometry.
A quantitative study of the development of type II pneumocytes in fetal lung
Authors: Leary J. F. , Finkelstein J. N. , Notter R. H. , Shapiro D. L. 
Published: August 2005

AbstractCell populations dissociated from fetal rabbit lungs were analyzed by laser flow cytometry for the presence of type II penumocytes. These cells are distinguishable by the staining of their lamellar bodies with the fluorescent lipophilic dye, phosphine‐3R and by their intensity of low‐angle light scatter. Lung cells were obtained by enzymatic dissociation from fetal rabbits at gestational ages of 24 d, 27 d, and from 2‐d newborn rabbits. Flow cytometric analysis was sufficiently sensitive to discriminate between fetuses. Quantitative analysis of type II pneumocytes showed that newborn rabbits had a distinct cell subpopulation in a region of low‐angle light scatter and phosphine‐3R fluorescence intensity similar to that previously reported on type II cells from adult rabbits. By contrast, 24‐d gestation rabbits had a negligible type II cell subpopulation. Fetuses of 27 and 30 d gestation showed a slow but progressive increase in the numbers of cells in the type II region. Mathematical analyses of light scatter and fluorescence intensity distributions were used to define statistically significant (P < .05) boundaries that characterize the development of the type II cell subpopulation in fetal rabbit lung. The methods employed offer new possibilities for quantification of developing lung cell subpopulations of particular interest to the problem of respiratory distress syndrome in human neonates.
A method for determining ploidy distributions in liver tissue by stereological analysis of nuclear size calibrated by flow cytometric DNA analysis
Authors: Danielsen Håvard , Lindmo Tore , Reith Albrecht 
Published: August 2005

AbstractA method is presented for determining ploidy distributions in mouse liver from image analysis with stereological estimations of nuclear size in tissue sections. Nuclear profile distributions obtained from profile measurements were subjected to a mathematical unfolding procedure in order to obtain the nuclear size distributions. Based on the assumption that nuclear size increases monotonically with nuclear DNA content, flow cytometric DNA analysis of suspensions of liver cell nuclei was used to calibrate the method, thus yielding the mean nuclear size of each ploidy class, i.e., diploid, tetraploid, and octaploid nuclei. After the size interval for each of the ploidy classes was determined, the method allowed determination of ploidy distributions in mouse liver by stereological image analysis alone.The method was established from combined stereological and flow cytometric measurements on liver tissue representing two different stages of liver regeneration after two‐thirds partial hepatectomy, and it was tested against an independent set of data representing a marked increase in the portion of S‐phase cells.
Dual‐laser, differential fluorescence correction method for reducing cellular background autofluorescence
Authors: Steinkamp John A. , Stewart Carleton C. 
Published: August 2005

AbstractA method has been developed for reducing the intrinsic autofluorescence background component in cells labeled with fluorescent antibodies, thus permitting low levels of antibody‐binding on highly autofluorescent cells to be quantified. The method is based on the broad autofluorescent excitation spectra compared to the well‐defined spectra of the fluorescent label. Two laser wavelengths were used, one optimally to excite the fluorescent label plus autofluorescence and the second to excite only the autofluorescence. Two fluorescence measurements were made in the same wavelength region and the signals were subtracted on a cell‐by‐cell basis using a difference amplifier to zero the autofluorescence and amplify the signal from the fluorescent label. Test results on unlabeled autofluorescent macrophages showed that the autofluorescence component was reduced by balancing the signal inputs to the difference amplifier. When labeled macrophages were analyzed, the autofluorescence was reduced and the fluorescent‐labeled antibody‐binding component was amplified. The method was also able to resolve labeled lymphocytes from unlabeled autofluorescent macrophages.
Mapping by chromosome sorting of several gene probes, including C‐myc, to the derivative chromosomes of a 3;8 translocation associated with familial renal cancer
Authors: Harris Peter , Morton Cynthia C. , Guglielmi Paul , Li Frederick , Kelly Kathleen , Latt Samuel A. 
Published: August 2005

AbstractIn eight members of a single family a constitutional translocation t(3;8) (p14.2;q24.1) is associated with the development of renal cancer. Chromosomes isolated from a cell line established from a subject with this translocation were analysed in flow with a fluorescence‐activated cell sorter (FACS II). Nearly six million chromosomes from the flow karyotype region containing the der(8) and 5.5 million from the region containing the der(3) were sorted, the DNA extracted, digested with EcoRI, size fractionated by electrophoresis, and transferred to nitrocellulose. Hybridization with gene probes for c‐mos, which has been localized to 8qll‐q22 and so‐matostatin, which has been mapped to 3q28, confirmed that the sorted fractions contained, respectively, the der(8) and der(3) chromosomes. The cellular oncogenes c‐raf‐1 (3p25) and c‐myc (8q24) were found to be translocated to the der(8) and der(3) chromosomes, respectively. The possible role that the relocation of c‐myc might have on the development of renal carcinoma in carriers of this 3;8 translocation was further studied by analysis of the region surrounding the c‐myc gene. By the use of cosmid cloning, no rearrangement 31 Kb 5′(or 19 Kb 3′) of the translocated gene was found, indicating that the breakpoint is not immediately adjacent to c‐myc.In an associated study, the DNA fragment D3S2 from chromosome 3 was found to map to 3p14.2‐pter. This assignment in conjunction with published somatic cell hybrid data enabled D3S2 to be mapped more precisely to the interval 3p14.2–3p21.
Extraction of nuclei from selected regions in paraffin‐embedded tissue
Authors: Oud Peter S. , Hanselaar Ton G. J. M. , Reubsaet‐Veldhuizen José A. M. , Meijer Jos W. R. , Gemmink Anita H. , Pahlplatz Martin M. M. , Beck Hans L. M. , Peter Vooijs G. 
Published: August 2005

AbstractA method for the extraction of nuclei from selected regions in paraffin‐embedded tissue is described. Fifty‐micrometer sections are cut, dewaxed, and rehydrated. For the final handling, the sections are manually transferred from one tray to another. The sections are put on a slide under a dissection microscope and the region of interest is isolated by scraping off the irrelevant region with a scalpel. An optimal number of single nuclei is obtained by incubation in a protease solution with intermediate syringing. The nuclei are washed and can be used for flow cytometry. Resuspension of the nuclei in foetal calf serum and cytocentrifugation results in preparations suited for image analysis. DNA cytometric measurements of nuclei in a carcinoma in situ and an invasive carcinoma region in breast tissue present in the same tissue block and in a severe dysplasia/carcinoma in situ (CIN III) region of the cervix are presented.
A simple method to select specific tumor areas in paraffin blocks for cytometry using incident fluorescence microscopy
Authors: van Driel‐Kulker A. M. J. , Eysackers M. J. , Dessing M. T. M. , Ploem J. S. 
Published: August 2005

AbstractA simple method is described for the selection of tumor areas in paraffin blocks for cytometry. The surface of a paraffin block is stained with the fluorescent dye DAPI. By means of incident fluorescence microscopy on the cut surface of a total block, the tissue can be visualized. Location of the tumor area with the aid of conventional histopathological criteria is feasible using the adjacent section after hematoxylin‐eosin staining as a diagnostic guideline. Once the position of the tumor area is determined, a small hollow bore, which is screwed in the objective holder, is pressed in the tissue. The depth is controlled by the microscrew of the microscope. After retraction of the bore, the block is removed, and a thick section is cut. The selected area can be processed for cytometry separately from the remaining tissue. The technique can be used either to enrich the sample to be analyzed with tumor cells or to analyze histopathologically different tumor compartments. Both flow and image cytometry can make use of this selection technique.
Representing cell cycle data
Authors: Ubezio Paolo , Capolongo Laura 
Published: August 2005

AbstractIn this communication we suggest two simple ways to represent the information on the cell cycle obtained by flow cytometry, offering some advantages over the traditional plots. We show that the results of a single experiment, when reduced to the percentages of cells in G1, S, and G2M phases, can be completely expressed with a single point in a G1–G2 plan where iso‐S lines are also drawn. By the use of this plot, the time course of the complete kinetics of a cell population may be shown in a single drawing. The second plot, we suggest, integrates information on cell cycle, represented by the distribution mean, with cell number.
A pragmatic approach to the analysis of DNA histograms with a definable G1 peak
Authors: Watson James V. , Chambers Stephen H. , Smith Paul J. 
Published: August 2005

AbstractA method for DNA histogram analysis is described that depends only on the simple assumption that the data are normally distributed and a requirement that a G1 peak is present. A probability density function was derived from the assumption that extracted the S‐phase component from the whole histogram. The model was tested with simulated data, and good agreement between predicted and known proportions in G1, S, and G1+M was found. Good agreement was also found between duplicates of experimentally derived data. Some systematic errors are present in the analysis of certain types of histograms. However, these result in small errors when compared with biological and experimental variation and are less than the average of algorithms in current use.The program required only two queued requests, those of the start and the end channels over which the analysis is to be performed. The algorithms perform rapidly on a micro‐computer with only 28K addressable memory. Only two failures occurred in over 350 analyses and the method can be used for drug‐ and radiation‐perturbed populations as well as with unperturbed.
Effects of hyperthermia, irradiation, and cytotoxic drugs on fluorescein isothiocyanate staining intensity for flow cytofluorometry
Authors: Dyson J. E. D. , McLaughlin J. B. , Surrey C. R. , Simmons Deena M. , Daniel Jill 
Published: August 2005

AbstractMeasurement of fluorescein isothiocyanate (FITC) staining intensity of cultured lymphoblastoid cells following hyperthermia showed large increases without concomitant increases in nuclear protein. Similar measurements of cells following incubation with cytotoxic drugs showed fluorescent intensity increases that exceeded the increases in nuclear protein that were due to the cell cycle blocking action of the drug. The reverse, however, was true for cells following irradiation. In contrast, FITC staining intensity and nuclear protein measurements of cells proceeding through the cell cycle after removal of the cycle blocking agent showed nearly parallel changes, although there were reproducible minor differences, especially following blocking with hydroxyurea. These results suggest that FITC staining intensity is a function not only of nuclear protein content but also of stain access to the reaction sites of the protein constitutents of the chromatin. Thus, it is possible that FITC staining may be used as a probe of changes in chromatin structure following experimental manipulation of cells in vitro or treatment of tumors in vivo.
A flow cytometric study of DNA staining in situ in exponentially growing and stationary Euglena gracilis
Authors: Bonaly J. , Bre M. H. , Lefort‐Tran M. , Mestre J. C. 
Published: August 2005

AbstractDNA stainability by different fluorochromes has been compared in exponentially dividing and stationary Euglena cells. With the intercalating fluorochromes, ethidium bromide, acridine orange and DAPI, a decrease of fluorescence intensity of the G1 cells is observed when cells enter stationary stage. However this decrease of fluorescence is not obtained with the nonintercalating fluorochrome Hoechst 33258. If nuclear basic proteins are extracted, however, the intensity of staining by either Hoechst 33258 or ethidiumbromide is comparable in stationary and dividing cells. Therefore, the decrease of fluorescence intensity of the G1 cells observed during the transition from exponential to stationary phase is not due to a loss of DNA but is related to the exposure of chromatin binding sites for ethidium bromide. In Euglena cells, DNA accessibility for intercalating fluorochromes depends upon chromatin structure and consequently upon cell age.
DNA flow cytometry of control Euglena and cell cycle blockade of vitamin B12‐starved cells
Authors: Lefort‐Tran M. , Bre M. H. , Pouphile M. , Manigault P. 
Published: August 2005

AbstractVitamin B12 starvation in Euglena induces a cell cycle arrest that leads to unbalanced growth. Microfluorometry and flow cytometry analyses of cellular DNA fluorescence after Hoechst 33258 staining were performed on control and vitamin B12‐deficient cells. Convergent results are obtained with both methods. Histograms that represent arrested cells are unimodal, with a mode channel value nearly twice that of the G1 control cell peak. Dispersion of fluorescence values is great, and values from 2C and over 4C are observed and discussed. It appears that vitamin B12 starvation in Euglena leads to defective DNA synthesis. Blocked cells have different DNA content, corresponding to blockade of DNA replication during the S phase. A second block prevents the onset of mitosis even for 4C cells.
Comparison of helium‐neon and dye lasers for the excitation of allophycocyanin
Authors: Loken Michael R. , Keij Jan F. , Kelley Keith A. 
Published: August 2005

AbstractAllophycocyanin (APC) has a broad absorption spectrum permitting several different lasers to be used to excite this dye in a flow cytometer. A comparison was made between a dye laser and a helium‐neon (HeNe) laser for the excitation of APC as an immunofluorescent chromophore. The ratio of fluorescence of stained to unstained lymphocytes (signal to background) was used to assess differences in sensitivity. In determining the best wavelength for operating the dye laser, it was found that there was little difference in the ability to separate the positive‐labelled cells from the unstained cells using 600 nm or 633 nm light for excitation of APC. A study of the effect of laser power on the signal to background identified a nonlinear relationship. It was found that the sensitivity obtained with 47 mW of 633 nm light from a HeNe laser was near the maximum attainable. This sensitivity was comparable to that obtained using phycoerythrin as an immunofluorescence chromophore. APC had the added advantage of being applicable to the study of highly autofluorescent cells. Exciting this chromophore using red light dramatically decreased the autofluorescence observed even on alveolar macrophages.
Closed loop television tracking of beating heart cells in vitro
Authors: Schuette W. H. , Burch C. , Roach P. O. , Parrillo J. E. 
Published: August 2005

AbstractThe motion of beating heart cells in vitro has been used as a sensitive indicator of the presence of drugs. The combination of closed loop television video tracking and latex microspheres placed on the cells as markers results in an improved measurement of cell motion. Latex microspheres, 5.2 micrometers, are allowed to settle onto beating my ocardial heart cells in vitro, where they move in concert with the cell motion. These microspheres, when viewed by televised transmission microscopy with normal optics, present high contrast circular targets that are excellent for closed loop television video tracking techniques. With video tracking, an analog voltage is generated proportional to the horizontal center of intensity of the target being tracked. This signal represents the location of a video demodulator gate that is maintained centered over the video target by feedback control.