bluuebunny/crossref_metadata_2025_split huggingface dataset:
TOTO and YOYO: New very bright fluorochromes for DNA content analyses by flow cytometry
Authors: Hirons G. T. , Fawcett J. J. , Crissman H. A. 
Published: June 2005

AbstractFlow cytometric (FCM) studies were performed on nuclei, ethanol‐fixed CHO cells, and isolated human GM130 chromosomes stained with two new cyanine dyes, TOTO and YOYO. These fluorochromes, which are dimers of thiazole orange and oxazole yellow, respectively, have high quantum efficiencies and exhibit specificities for both DNA and RNA. Bound to dsDNA in solution, TOTO and YOYO emit at 530 and 510 nm, respectively, when excited at 488 nm and 457 nm, wavelengths available from most lasers employed in FCM. RNase‐treated CHO nuclei, stained with either TOTO or YOYO, provided DNA histograms, with low coefficients of variation, that were as good as or better than those obtained with nuclei stained with propidium iodide (PI) or mithramycin (MI). In addition, by comparison on an equimolar basis, nuclei stained with YOYO fluoresced over 1,000 times more intensely than nuclei stained with MI. Fluorescence ratio analyses of nuclei stained with both YOYO and Hoechst 33258 showed that the ratio of YOYO to Hoechst fluorescence remained relatively constant for G1 and S phase cells, but decreased significantly for cells in G2/M. These results indicate that the cyanine dyes may be useful in examining specific changes in chromatin structure during G2/M phases of the cell cycle. Ethanol‐fixed CHO cells stained with TOTO or YOYO did not yield reproducible DNA histograms of good quality, presumably because of the poor accessibility of DNA to these large fluorochromes. However, bivariate analyses of human GM130 chromosomes stained with TOTO or YOYO alone and excited sequentially with uv and visible wavelengths showed resolution of many individual chromosome peaks similar to results obtained for chromosomes stained with HO and chromomycin A3. Collectively, these studies show potential advantages for the use of these new cyanine dyes in FCM studies that require the sensitive detection of DNA. © 1994 Wiley‐Liss, Inc.
Discrimination of bromodeoxyuridine labelled and unlabelled mitotic cells in flow cytometric bromodeoxyuridine/DNA analysis
Authors: Jensen Peter Østrup , Larsen Jørgen K. , Christensen Ib Jarle , van Erp Piet E. J. 
Published: June 2005

AbstractBromodeoxyuridine (BrdUrd) labelled and unlabelled mitotic cells, respectively, can be discriminated from interphase cells using a new method, based on immunocytochemical staining of BrdUrd and flow cytometric four‐parameter analysis of DNA content, BrdUrd incorporation, and forward and orthogonal light scatter. The method was optimized using the human leukemia cell lines HL‐60 and K‐562. Samples of 105 ethanolfixed cells were treated with pepsin/HCl and stained as a nuclear suspension with anti‐BrdUrd antibody, FITC‐conjugated secondary antibody, and propidium iodine. Labelled mitoses could be discriminated from unlabelled mitoses, and from labelled and unlabelled G2 cells, by their intermediate log FITC fluorescence intensity. In addition, mitoses and G2 nuclei differed in forward and orthogonal light scattering, but had equal intensity of propidium iodide fluorescence. This method for discrimination of labelled mitoses was also tested on cultured normal adult human keratinocytes labelled with iododeoxyuridine (IdUrd). In keratinocytes, where the cell structure was preserved after pepsin/HCl, IdUrd labelled mitotic cells were similarly discriminated in the log FITC/propidium iodide fluorescence distribution. This interpretation was supported by experiments using mitotic arrest, fluorescence activated cell sorting and microscopy, and comparison with an alternative flow cytometric method for discrimination of mitoses. © 1994 Wiley‐Liss, Inc.
Detection of genetic changes in Barrett's adenocarcirioma and Barrett's esophagus by DNA in situ hybridization and immunohistochemistry
Authors: Krishnadath K. K. , Tilanus H. W. , Alers J. C. , Mulder A. H. , van Dekken H. 
Published: June 2005

AbstractWe have investigated the occurrence of chromosomal DNA and cell cycle‐related protein changes in Barrett's epithelium and adenocarcinoma. The presence of numerical chromosomal aberrations was studied by applying nonisotopic in situ hybridization (ISH) with (peri‐)centromeric DNA probes, specific for chromosomes 7, 8, 17, and Y, to routinely processed tissue sections of five cases (4 male, 1 female) of Barrett's adenocarcinoma and adjacent Barrett's epithelium. Cell cycle‐related protein expression was studied by immunohistochemistry (IHC) for p53 protein and the Ki‐67 antigen (Mib‐1) in subsequent sections. P53 protein overexpression was found in 3 of the 5 tumors. Overrepresentation of chromosome 8 and loss of chromosome 17 were found in 2 adenocarcinomas, both also negative for p53 protein overexpression. Y‐loss, mostly clonal, was detected in 3 of the 4 male adenocarcinomas and 2 cases of adjacent Barrett's epithelium. One tumor had both areas of overrepresentation and loss of the Y chromosome. All Barrett's adenocarcinomas appeared to contain aneuploid cell populations. No relation was found between cell proliferation characteristics and chromosomal aberrations. We conclude that ISH with chromosome specific DNA probes can be applied for the assessment of potentially important numerical chromosome changes in Barrett's esophagus. Further, the combination of IHC and ISH is useful for evaluation of specific genetic events. © 1994 Wiley‐Liss, Inc.
Automated image cytometry for detection of rare, viral antigen‐positive cells in peripheral blood
Authors: Ploem‐Zaaijer Joke J. , Mesker Wilma E. , Boland Greet J. , Sloos Willem C. R. , van de Rijke Frans M. , Jiwa Mehdi , Raap Anton K. 
Published: June 2005

AbstractA cell detection method based upon automated screening is described for recognition of low frequencies (1 in 100,000) of immuno‐enzymatically labelled white blood cells in human peripheral blood. The used image cytometry instrumentation (LEYTAS) includes a wide‐field, fully automated microscope (Autoplan) and a modular image analysis computer (MIAC), both from Leica, Wetzlar, Germany. The MIAC contains image boards for optimum use of mathematical morphology algorithms. Communication with the MIAC is via a personal computer. Programs for automated cell analysis have been written in C language. Main features of the system are fast analysis of, large microscope fields including a count of all cells, selection of objects of interest (alarms), and display of digitally stored images of these alarms. We tested this system for the detection of white blood cells expressing antigen of cytomegalovirus (pp65) in 50 human blood smears from kidney transplant recipients. Immuno‐enzymatic (peroxidase) staining was performed with DAB and counterstaining with hematoxylin. For determination of the sensitivity, a series of dilutions of a positive sample with a negative sample was performed. The lowest frequency detected was 1 antigenpositive cell / 3 × 105 antigen‐negative cells. Screening time was about 60 min for one million cells. © 1994 Wiley‐Liss, Inc.
Flow cytometric analysis of bioluminescence emitted by recombinant baculovirus‐infected insect cells
Authors: Lindqvist Christer , Karp Matti , Åkerman Karl , Oker‐Blom Christian 
Published: June 2005

AbstractFive recombinant baculoviruses, each containing a different insect luciferase gene encoding a protein with characteristic light emission properties, namely, luc GR (546 nm), luc FF (556 nm), luc YG (560 nm), luc YE (578 nm), and luc OR (593 nm) were constructed. All genes were inserted under the transcriptional control of the polyhedrin gene promoter of the Autographa californica nuclear polyhedrosis virus (AcNPV) and expressed in Spodoptera frugiperda insect cells during viral infection. The biological activity of the different luciferases was characterized by using intact recombinant baculovirus infected cells. Addition of the substrate, D‐luciferin, immediately prior to the analysis allowed monitoring of light emission by flow cytometry. Also, the kinetics of the light emission of lucGR was analyzed with the flow cytometer. The emission peaks of the infected cells were clearly separated by wavelength scanning. Especially, the firefly luciferase (lucFF) had a broad peak and transient luminescence. The highest maximal intensity values in vivo were recorded for luc GR and luc YG. SDSPAGE analysis showed that the major protein expressed had a molecular weight similar to authentic luciferase. Flow cytometry and insect luciferases with clearly separated emission spectra appear to be of value for sensitive in vivo analysis of gene promoter activity. © 1994 Wiley‐Liss, Inc.
Flow cytometric analysis of entire microbial colonies
Authors: Sahar Eli , Nir Raphael , Lamed Raphael 
Published: June 2005

AbstractMuch could be gained if the scope of flow cytometry could be broadened to the study of entire cell colonies, rather than to populations of single, separate cells. This can be achieved by encapsulating single microbial cells in small spheres in a way that allows each cell to multiply and form a colony within its respective microbead, which is then amenable to analysis by flow cytometry. Methods for performing the encapsulation within beads of appropriate size and homogeneity have been developed (Nir et al., Appl. Environ. Microbiol. 56:2870–2875, 1990). We describe here how a variety of properties of the entrapped colonies, such as mass, growth rate, enzymatic activity, and expression of specific antigens, can be measured, and we discuss how these constructs can be utilized to select microbial mutants. © 1994 Wiley‐Liss, Inc.
Further flow cytometric studies of the effects of triethylenemelamine on somatic and testicular tissues of the rat
Authors: Bickham John W. , Sawin Virginia L. , McBee Karen , Smolen Michael J. , Derr James N. 
Published: June 2005

AbstractExposure to the mutagen triethylenemelamine on rat bone marrow, blood, and testis was studied using flow cytometry of DAPI‐stained nuclei. Increased coefficients of variation (CVs) of the G1 peaks were observed in bone marrow and blood after both 1 d and 5 d exposures. After 5 d exposure and 7 d recovery both tissues had recovered, in some cases to significantly lower CVs. Increased CVs of the 1C peak of testis were observed only after 5 d exposure to the high dose with, no subsequently observed recovery. Bone marrow cells also were stained with, Hoechst 33258 and Propidium Iodide. No differences among dyes were observed indicating that increased CVs likely are due to DNA damage resulting from interactions with the mutagen rather than differences in how the dyes bind to DNA relative to mutagen binding. This study demonstrates that differences occur among tissues in how quickly they respond and recover from mutagen exposure. Increased CVs, cell cycle alterations, and decreased CVs after recovery are all potentially useful biomarkers of effect for laboratory and field studies in environmental toxicology. © 1994 Wiley‐Liss, Inc.
Detection of human papillomavirus type 16/18 DNA in cervicovaginal cells by fluorescence based in situ hybridization and automated image cytometry
Authors: Siadat‐Pajouh Majid , Periasamy Ammasi , Ayscue Andrea H. , Moscicki Anna B. , Palefsky Joel M. , Walton Leslie , Demars Leslie R. , Power Jon D. , Herman Brian , Lockett Stephen J. 
Published: June 2005

AbstractAutomatic fluorescence image cytometry (AFIC) is a fast, sensitive, and reliable approach for screening slide‐based clinical specimens. In this study, we applied AFIC to identify cancer‐associated human papillomavirus (HPV) genotypes 16 and 18 in individual cells of cervical smears using a sensitive fluorescence based in situ hybridization (FISH) assay. HPV sequences were labeled by FISH and the cells imaged using an epi‐fluorescence microscope coupled to a low‐light color CCD camera. Before application to clinical specimens, AFIC was assessed using fluorescent calibration beads and cervical cancer cell lines containing known numbers of integrated HPV genomes per nucleus. Assessment showed that our AFIC had a linear response, was quantitatively accurate, and had the sensitivity to detect one HPV genome per nucleus: After acquisition of images, computer algorithms identified every cell nucleus (via a fluorescent DNA counterstain) and quantified the FISH signal per nucleus. AFIC was employed to screen 27 patient specimens for HPV 16/18, of which 12 were positive. The HPV status of the specimens positively correlated with the pathological diagnosis, and since AFIC automatically and correctly located every cell, it was possible to directly compare morphology and HPV status in the same cell. In conclusion, the combination of FISH and AFIC is a sensitive and quantitative method to detect high risk HPV sequences in cervical smears. © 1994 Wiley‐Liss, Inc.
Lymphocyte subset analysis by boolean algebra: A phenotypic approach using a cocktail of 5 antibodies and 3 color immunofluorescence
Authors: Hunter Stephen D. , Peters Lyndsay E. , Wotherspoon John S. , Crowe Suzanne M. 
Published: June 2005

AbstractCommercial reagent kits for the evaluation of leukocyte subsets involve the staining of a panel of up to six tubes using combinations of pre‐mixed fluorescein isothiocyanate (FITC) and R‐phycoerythrin (PE) conjugated, monoclonal antibodies. We describe a rapid method whereby total CD3+ T‐cells, CD4+ T‐cells (CD3+ CD4+), CD8+ T‐cells (CD3+CD8+), putative γδ‐receptor‐T‐cells (CD3+CD4−CD8−), and T‐cells that are CD3+ CD4+ CD8+ as well as B‐lymphocytes and NK‐cells can be enumerated after staining in a single tube. Whole blood specimens are labelled with a mixture of antibodies: FITC‐conjugated antibodies to CD4 and CD19, PE‐conjugated antibodies to CD8 and CD16, and either peridinin chlorophyll protein (PerCP) or allophycocyanin (APC) labelling for antibodies to CD3. After recording 20,000 events the data were analysed on the Consort 32 computer system and LYSYS‐II (Becton Dickinson, San Jose, CA) and all of the lymphocyte subset values were determined by Boolean algebra using a technique we refer to as Boolean gate analysis (BGA). Our study has shown that BGA is statistically equivalent to SimulSET lymphocyte subset analysis. Furthermore, the procedure reduces the number of tubes required to two with consequential saving in reagents, consumables, and time. © 1994 Wiley‐Liss, Inc.
Visible diode lasers can be used for flow cytometric immunofluorescence and DNA analysis
Authors: Doornbos Richard M. P. , de Grooth Bart G. , Kraan Yvonne M. , van der Poel Carel J. , Greve Jan 
Published: June 2005

AbstractThis report describes a feasibility study concerning the use of a visible diode laser for two important fluorescence applications in a flow cytometer. With a 3 mW 635 nm. diode laser, we performed immunofluorescence measurements using the fluorophore allophycocyanin (APC). We have measured CD8 positive lymphocytes with a two‐step labeling procedure and the resulting histograms showed good separation between the negative cells and the dim and the bright fluorescent subpopulations. As a second fluorescence application, we chose DNA analysis with the recently developed DNA/ RNA stains TOTO‐3 and TO‐PRO‐3. In our setup TO‐PRO‐3 yielded the best results with a CV of 3.4%. Our results indicate that a few milliwatts of 635 nm light from a visible diode laser is sufficient to do single color immunofluorescence measurements with allophycocyanin and DNA analysis with TO‐PRO‐3. The major advantages of using a diode laser in a flow cytometer are the small size, the low price, the high efficiency, and the long lifetime. © 1994 Wiley‐Liss, Inc.
Simultaneous cytokinetic measurement of aneuploid tumors and associated diploid cells following continuous labelling with chlorodeoxyuridine
Authors: White R. Allen , Pollack Alan , Terry Nicholas H. A. 
Published: June 2005

AbstractCells from a murine tumor, MCa‐K, were continuously labelled with the thymidine analogue chlorodeoxyuridine (CldUrd) and analyzed by bivariate flow cytometry in order to measure the growth fraction (GF) and potential doubling time (T pot) of both the DNA‐aneuploid tumor cells and the associated DNA‐diploid cells. MCa‐K has a DNA index of 1.7, rendering two, partially overlapping, populations observable with labelled and unlabelled cells in each population. The data from these tumors may be divided into three regions of differing DNA content, with one region containing a pure DNA‐diploid population, a second region with both cell types, and a third region including only DNA‐aneuploid cells. Equations are presented to characterize the fractions of labelled cells in each region as a function of labelling time and cell type, thereby permitting estimation of the proliferative properties of the populations. These equations include the possibility that DNA‐aneuploid cells cease cycling both in G1 and in S phase to account for the observed numbers of unlabelled cells with S phase contents. The estimated value of Tpot of the DNA‐diploid cells is 126.0 h with a GF of 42%, while that of the DNA‐aneuploid cells is 36.9 h with a GF of 69%. It is also estimated that between 2% and 6% of all DNA‐aneuploid cells starting DNA synthesis cease cycling, leading to 25% of the cells having an S‐phase DNA content being noncycling. © 1994 Wiley‐Liss, Inc.
Evaluation of methods for measuring cellular glutathione content using flow cytometry
Authors: Hedley David W. , Chow Sue 
Published: June 2005

AbstractThe currently available flow cytometric stains for cellular glutathione were evaluated, examining the labelling of both human and rodent cell lines under various conditions of concentration, time, and temperature. Procedures were used that depleted, glutathione (GSH) while having a minimal effect on other cellular sulphydryls in order to estimate linearity and the extent of background staining. As previously reported, monochlorobimane was highly specific for GSH in rodent cells but failed to label human cells adequately because of its low affinity for human glutathione S‐transferases. Higher concentrations of monochlorobimane achieved more complete labelling of, the human cellular GSH pool but gave increased background fluorescence due to non‐GSH binding. The analogue monobromobimane, which binds nonenzymatically to sulphydryls, reacted, more readily with GSH than with protein sulphydryls and, provided that stain concentration and incubation time were controlled, gave reproducible staining of human cells with 20% of total fluorescence due to background staining. Of the currently available stains for measuring GSH in human cells, monobromobimane is the agent of choice. Mercury orange also binds more readily to GSH than to protein, giving a degree of specificity, and it has the additional advantage of being excited at 488 nm. However, the reproducibility of staining with mercury orange was less consistent than that using monobromobimane, and a higher background fluorescence was seen. Two additional stains, O‐phthaldialdehyde and chloromethyl fluorescein, could also be used to label cellular GSH, but both gave an unacceptably high level of background staining. It is recommended that flow cytometric GSH assays should routinely include a sample of cells that have been depleted of GSH in order to determine the extent of background labelling. © 1994 Wiley‐Liss, Inc.
Detection of intracellular HIV in lymphocytes by flow cytometry
Authors: Gadol Nancy , Crutcher Gillian J. , Busch Michael P. 
Published: June 2005

AbstractVarious permeabilization procedures and defined gating techniques were tested in order to optimize existing flow cytometric assays and devise a specific assay for the direct detection of intracellular HIV‐1 antigens in clinical blood specimens. In our optimal procedure, blood lysed with Orthomune Lysing Reagent was fixed with 3.7% formaldehyde for 10 min at room temperature and then permeabilized with 0.2% Tween 20 for 15 min at room, temperature. Cells from whole blood were labeled with either FITC‐anti‐p18 or FITC‐anti‐p24 monoclonal antibodies and PE‐anti‐Leu M9 (CD33) in order to exclude monocytes and granulocytes from the lymphocyte gate. The assay demonstrated that mean percentages of HIV p24 antigen positive cells were increased in patients with advanced disease. The assay in its present form is useful for monitoring disease progression and for monitoring the effects of antiviral therapy in individuals, but it is not currently sensitive enough to detect consistently the low levels of HIV infected peripheral blood cells in asymptomatic individuals. © 1994 Wiley‐Liss, Inc.
Automatic fluorescence metaphase finder speeds translocation scoring in FISH painted chromosomes
Authors: Piper J. , Poggensee M. , Hill W. , Jensen R. , Ji L. , Poole I. , Stark M. , Sudar D. 
Published: June 2005

AbstractA fluorescence metaphase finder was constructed with commercially available hardware and a standard Unix workstation. Its accuracy was measured in terms of the number of false positive and false negative detected metaphases on a variety of different slide preparations. The metaphase finder was used in a translocation scoring experiment in which metaphase preparations of human peripheral blood lymphocytes were hybridized with whole chromosome probes to chromosomes #1, #2, and #4. The automatic finder presented metaphases to the cytogeneticist, centered in the eyepieces at × 63. The cytogeneticist's scores of analyzable metaphases and of painted chromosomes involved in rearrangements were recorded. The time for the analysis was recorded and compared to the time to analyze a similar number of cells in a purely visual experiment in which the cytogeneticist scanned for cells and analyzed them, both at × 63. The results showed that, neglecting the machine time spent scanning unattended, the amount of time required for the analysis was reduced by a factor of three. Furthermore, in this experiment the metaphase finder found more scorable metaphases than the cytogeneticist found by visual scanning. Machine‐assisted scoring had additional, less quantifiable, benefits; notably that digital images of metaphases sometimes assisted the analysis of chromosome rearrangements, that cells could be revisited easily, and that the analysis was much less fatiguing. © 1994 Wiley‐Liss, Inc.
Flow‐cytometric analysis of apoptotic and nonapoptotic T‐cell receptor‐transgenic thymocytes following in vitro presentation of antigen
Authors: Curnow S. J. , Barad M. , Brun‐Roubereau N. , Schmitt‐Verhulst A.‐M. 
Published: June 2005

AbstractThe use of flow cytometry to detect apoptotic thymocytes is now well established. We have further developed the technique of Hoechst 33342 vital staining to identify discrete stages of murine thymocyte apoptosis (induced by 37°C culture), in conjunction with propidium iodide (PI), cell scatter profile, and surface marker analysis. The first detectable stage was an increase in Hoechst fluorescence without any change in plasma membrane permeability (measured by PI staining). At this early stage thymocytes had already reduced in size, fragmented their DNA, and for the predominant CD4+ CD8+ double positive population, reduced expression of CD4 and CD8. Subsequent to this stage thymocytes continued to reduce in size and decrease expression of CD4 and CD8, though this was accompanied by an increase in membrane permeability. This technique was applied to an in vitro antigen‐specific deletion system, where apoptosis of T cell‐receptor‐transgenic thymocytes was induced upon presentation of self‐antigen. Although self‐antigen‐induced apoptotic thymocytes showed similar characteristics to those undergoing spontaneous apoptosis, there was a significant population of nonapoptotic CD4+8+ thymocytes that also had reduced expression of CD4 and CD8. Therefore, we have been able to show that the reduced expression of CD4 and CD8 is not limited to apoptotic thymocytes. © 1994 Wiley‐Liss, Inc.