bluuebunny/crossref_metadata_2025_split huggingface dataset:
Adaptation of the TdT assay for semi‐quantitative flow cytometric detection of DNA strand breaks
Authors: Bromidge Teresa J. , Howe Denise J. , Johnson Stephen A. , Phillips Malcolm J. 
Published: June 2005

AbstractThe enzyme Terminal Deoxynucleotidyl Transferase (TdT) is a DNA polymerase which can be used to label DNA strand breaks by the incorporation of a labelled nucleotide followed by a fluorescent detection step. The amount of label incorporated can then be assessed by flow cytometry. The mechanism of action of TdT, however, will allow the addition of varying numbers of nucleotides to the free 3' termini produced by DNA strand breaks. The substitution of Digoxigenin (DIG)TM labelled dideoxynucleotides for labelled deoxy‐nucleotides in the TdT assay will limit the addition of label to a DNA break to a single nucleotide, thus ensuring a direct relationship between an increase in DNA strand breaks and an increase in fluorescence. We have used this adaptation of the TdT Assay to evaluate DNA damage incurred in lymphocytes, from patients with Chronic Lymphocytic Leukaemia (CLL), on exposure to UV irradiation and apoptosis‐inducing drugs, fludarabine and 2‐Chloro‐2′‐deoxyadenosine (2‐CdA). This technique may give a good indication of the susceptibility of CLL patients to apoptosis inducing drugs, and hence an indication of the likely response to these therapies. © 1995 Wiley‐Liss, Inc.
Photomultiplier voltage setting: Possible important source of variability in molecular equivalents of soluble fluorochrome (MESF) calculation?
Authors: Rigolin Gian Matteo , Lanza Francesco , Castoldi Gianluigi 
Published: June 2005

AbstractWe evaluated the effect of flow cytometer photomultiplier (PMT) voltage setting on molecular equivalents of soluble fluorochrome (MESF) calculation by using two different quantitative microbead calibrating kits as calibrating standards and a third one as an unknown testing sample of stable intensity of fluorescence. Based on the analysis of residual values derived from linear regression and on the determinations of the resolution index, we demonstrated that, on a FACScan instrument, the window of photomultiplier setting for fluorescein isothiocyanate (FITC) fluorescence determinations is 590–670 V. However, the best region was in the range of 610–650 V. Within the 590–670 PMT voltage region, the evaluation of the same testing sample at different PMT voltages, at 590 V, gave MESF percentage differences of 15% and 13% on the two lower unknown standards of intensity compared to the values obtained at the nearby 630 V setting. In the analyzed PMT region, logarithmic amplifier response was globally maintained, whereas evaluation of the same unknown testing sample in both linear (Lin) and logarithmic (Log) amplification demonstrated (at the higher intensities of fluorescence) Log‐Lin minimal differences within the 610–650 PMT voltage range. According to our data, it can be stated that PMT voltages between 610 and 650 provide the best instrument performances and that PMT setting may be proposed as a source of variability in MESF calculation that deserves a careful evaluation in quality‐control trials. © 1995 Wiley‐Liss, Inc.
Nucleic acid dyes for detection of apoptosis in live cells
Authors: Frey Tom 
Published: June 2005

AbstractApoptotic thymocytes were found to be much dimmer than normal thymocytes when stained with several nucleic acid dyes. These dyes provide a quick and simple assay for apoptosis which works for live cells and does not require a UV laser. The collection of dyes giving this staining pattern includes reagents suitable for use in either the FL1, FL2, or FL3 channel of a standard FACScan. Cells identified by these reagents were identical to apoptotic thymocytes defined by several widely used criteria: (i) rapid uptake of Hoechst 33342 but exclusion of propidium iodide, (ii) merocyanin 540 bright, and (iii) sub‐G1 DNA content when permeabilized in a buffer that elutes fragmented DNA. In addition, L3T4/Thy‐1 dim thymocytes were included in the dye dim population. The standard Hoechst 33342 and merocyanin 540 assays were not able to separate the normal and apoptotic populations in HL‐60 cells treated with camptothecin. However, the dyes SYTO‐16 and LDS‐751 both gave adequate differentiation of apoptotic from nonapoptotic cells in this model system. Some of these dyes also emit very little in other fluorescence channels of the flow cytometer and can be used in multicolor assays on cytometers equipped with only a single argon‐ion laser. © 1995 Wiley‐Liss, Inc.
Kinetics of plasma membrane and mitochondrial alterations in cells undergoing apoptosis
Authors: Lizard Gérard , Fournel Sylvie , Genestier Laurent , Dhedin Nathalie , Chaput Christophe , Flacher Monique , Mutin Mireille , Panaye Geneviève , Revillard Jean‐Pierre 
Published: June 2005

AbstractProgrammed cell death or apoptosis is characterized by typical morphological alterations. By transmission electron microscopy, apoptotic cells are identified by condensation of the chromatin in tight apposition to the nuclear envelope, alteration of the nuclear envelope and fragmentation of the nucleus, whereas integrity of the plasma membrane and organelles is preserved. Conversely cells undergoing necrosis display an early desintegration of cytoplasmic membrane and swelling of mitochondria. In this study we assessed by flow cytometry the sequential alterations of forward angle light scatter, 90° light scatter, and fluorescence associated with fluorescein diacetate, rhodamine 123, and propidium iodide in two human B cell lines undergoing apoptosis induced by the topoisomerase II inhibitor VP‐16. The kinetics of these modifications were compared to those of cells undergoing necrosis induced by sodium azide. At the same time intervals, cells were examined by transmission electron microscopy and by UV microscopy after staining with Hoechst 33342. We report that sequential changes in light scatters and fluorescein diacetate are similar in cells undergoing apoptosis or necrosis, whereas apoptosis is characterized by a slightly delayed decrease of mitochondrial activity as assessed by rhodamine 123 staining. Surprisingly a part of cells undergoing apoptosis displayed an early uptake of propidium iodide followed by a condensation and then a fragmentation of their nuclei. It is concluded that uptake of propidium iodide is a very early marker of cell death which does not discriminate between necrosis and apoptosis. Along with biochemical criteria, nuclear morphology revealed by staining with Hoechst 33342 would seem to be of the most simple and most discriminative assay of apoptosis. © 1995 Wiley‐Liss, Inc.
Rhodamine 123: A useful probe for monitoring T cell activation
Authors: Ferlini Cristiano , Biselli Roberto , Nisini Roberto , Fattorossi Andrea 
Published: June 2005

AbstractThe T cell activation pathway involves an increase in mitochondrial activity. This can be evaluated in individual cells using the fluorescent probe rhodamine 123 (Rh123) and flow cytometry. Peripheral blood mononuclear cells (PBMC) were stimulated with optimal concentrations of phytohaemagglutinin(PHA), superantigens (Sag) SEA and SEC2, and allogeneic cells. Activation kinetics were followed at days 1, 2, 4 and 7. In all activation conditions, Rh123 uptake was augmented with the CD25 expression, cell size, and DNA synthesis. Rh123 uptake reflected an increase in mitochondrial activity and mass, as assessed by experiments in which Rh123 was substituted for by the 10‐nonyl acridine orange, which stains mitochondria in an energy‐independent manner. The spectral characteristics of Rh123 allowed us to double stain cells with Rh123 and phycoerythrin‐conjugated monoclonal antibodies. In PHA‐activated cultures, CD4+ and CD8+ cells incorporated essentially the same amount of Rh123 at all time points, suggesting that the two subsets did not differ in their activation kinetics. Accordingly, after 1 weekof culture, no significant modification in the Cd4/Cd8 ratio was observed. Sag‐activated CD4+ cells incorporated a higher amount of Rh123 than did CD8+ cells and preferentially expanded after 1 week of culture as indicated by the increase in the CD4/CD8 ratio. The different behavior of the CD4 and CD8 subsets observed by dual color flow cytometry in the PHA and Sag models was confirmed using purified CD4+ and CD8+ cell preparations obtained by immunomagnetic sorting. CD4+ cells were also the preferential target in the allogeneic model, although the magnitude of the phenomenon was lower than in the Sag model. Present data indicate that Rh123 is a reliable marker for monitoring the mitochondrial compartment during T cell activation. The possibility of phenotyping Rh123‐stained cells adds to the applicability of the probe. © 1995 Wiley‐Liss, Inc.
Use of light scatter when recording a DNA histogram from paraffin‐embedded tissue
Authors: Ormerod M. G. , Titley J. C. , Imrie P. R. 
Published: June 2005

AbstractThe quality of a DNA histogram as recorded in a flow cytometer can often be improved by gating on forward and orthogonal light scatter. This may be helpful when measuring DNA histograms from formalin‐fixed, paraffin‐embedded material. We report that nuclei from many human tumours scatter more light orthogonally than those from normal stroma. Gating on light scatter enabled the DNA histogram from the tumour to be recorded with reduced contamination from normal cells. In studies of human mammary carcinomas, we have found that this method has the potential to improve the estimation of S phase and the measurement of DNA index (DI) when the DI is close to 1 (near diploid). © 1995 Wiley‐Liss, Inc.
Simultaneous dual‐frequency phase‐sensitive flow cytometric measurements for rapid identification of heterogeneous fluorescence decays in fluorochrome‐labeled cells and particles
Authors: Deka Chiranjit , Cram L. Scott , Habbersett Robert , Martin John C. , Sklar Larry A. , Steinkamp John A. 
Published: June 2005

AbstractIn frequency‐domain lifetime spectroscopy, the apparent fluorescence lifetimes obtained from phase‐shift measurements are independent of modulation frequency only in the special case of a single exponential fluorescence decay. For heterogeneous fluorescence decay, the apparent fluorescence lifetimes measured by the phase‐shift methods are functions of the modulation frequency. This modulation‐frequency dependent property of apparent fluorescence lifetimes may be used to identify heterogeneous fluorescence decays by measuring lifetimes at multiple frequencies. In this article we explore the requirements and experimental design considerations for making such measurements in flow. We report a phase‐sensitive flow cytometric method that allows one to probe the excited statelifetimes of labeled cells by using multiple simultaneous modulation frequencies. Application of this method is demonstrated by measuring fluorescence lifetimes of labeled cells at two frequencies simultaneously, using a continuous‐wave, dual‐frequency modulated excitation in flow. The dual‐frequency method presented herein can be used to rapidly identify heterogeneity in the fluorescence decay on a cell‐by‐cell basis in real time. Information on the nature of the fluorescence decay is important in biological measurements because it can provide insight into intermolecular interactions at the subcellular level.
Changes in free cholesterol content, measured by filipin fluorescence and flow cytometry, correlate with changes in cholesterol biosynthesis in THP‐1 macrophages
Authors: Hassall David G. , Graham Annette 
Published: June 2005

AbstractThe free cholesterol content of cells can be monitored by the intensity of fluorescence emissions from the polyene antibiotic filipin. In a previous study (Hassall: Cytometry 13:381–388, 1992) using THP‐1 macrophages, a decrease in filipin fluorescence in response to increasing concentrations of modified lipoprotein was observed, suggesting a reduction in the free cholesterol content of the cells. In this study, THP‐1 macrophages were treated with a number of agents known to modulate cholesterol biosynthesis and cholesterol esterification. Changes in filipin fluorescence emissions were measured by flow cytometry, and correlated with changes in cholesterol biosynthesis measured by incorporation of [14C]acetate into cholesterol. A correlation between decreases in filipin fluorescence and reductions in cholesterol biosynthesis was apparent, even when cholesterol esterification was inhibited. These results suggest that the decreases in filipin fluorescence observed may be due, at least in part, to reductions in cholesterol biosynthesis. © 1995 Wiley‐Liss, Inc.
Flow sorting of the Y sex chromosome in the dioecious plant Melandrium album
Authors: Veuskens Jacky , Marie Dominique , Brown Spencer C. , Jacobs Michel , Negrutiu Ioan 
Published: June 2005

AbstractThe preparation of stable chromosome suspensions and flow cytometric sorting of both the Y sex chromosome of the white campion, Melandrium album, and the deleted Y chromosome of an asexual mutant, 5K63, is described. The principle has been to maintain transformed roots in vitro, synchronise and block mitosis, reduce cells to protoplasts, and lyse these to release chromosomes. Such in vitro material, unlike many cell suspensions, showed a stable karyotype. Factors critical to producing high‐quality chromosome suspensions from protoplasts include osmolality of isolation solutions and choice of spindle toxin and of lysis buffer. Agrobacterium rbizogenes transformed young growing root cultures were synchronised at G1/S with 50 μM aphidicolin for 24 h and released to a mitotic block with 30 μM oryzalin for 11 h. Protoplast preparations from such tissue routinely had metaphase indices reaching 15%. Suspensions of intact metaphase chromosomes, with few chromatids, were obtained by lysing swollen mitotic protoplasts in a citric acid/disodium phosphate buffer. Except for the presence of clumps of autosomal chromosomes near the X and Y chromosome zones, monoparametric histograms of fluorescence intensities of suspensions stained with 4′,6‐diamidino‐2‐phenylindole showed profiles similar to theoretical flow karyotypes. Two types of Y chromosomes, one full‐length and one partially deleted (from the asexual mutant), could be sorted at 90% purity (21‐fold enrichment of Y). These results are discussed in the context of sex determination and differentiation in higher plants. © 1995 Wiley‐Liss, Inc.
Evidence of a selective depletion of a CD16+ CD56+ CD8+ natural killer cell subset during HIV infection
Authors: Lucia Barbara , Jennings Cheryl , Cauda Roberto , Ortona Luigi , Landay Alan L. 
Published: June 2005

AbstractThree‐color flow cytometric analysis of CD16+ natural killer (NK) cells was assessed in HIV seropositive patients and healthy heterosexual controls. A selective depletion of lymphocytes with the CD16+ NK phenotype was found among the HIV+ infected patients. When the CD16 lymphocyte subset was further evaluated by three‐color flow cytometry, cells bearing both the CD8 and CD56 antigens were significantly decreased. Analysis of activation antigens revealed a large proportion of CD16+ NK cells from HIV+ patients expressed HLA‐DR, but this did not correlate with CD25 (IL‐2 receptor) expression. The overall loss of the CD8 and CD56 antigens among the NK population with an increase in activation status may be due to differential loss of the NK cell subsets or, alternatively, to the loss of immunoregulatory cytokines, which have been shown to be important in maintaining NK activity. Whether these changes in the NK compartment may influence the outcome of individuals with HIV disease still remains an open question but is an important issue when performing phenotypic analysis of HIV+ subjects. © 1995 Wiley‐Liss, Inc.
Flow cytometric reticulocyte maturity index: A useful laboratory parameter of erythropoietic activity in anemia
Authors: Davis Bruce H. , Ornvold Kim , Bigelow Nancy C. 
Published: June 2005

AbstractFlow cytometric reticulocyte analysis is superior to manual reticulocyte counting with respect to precision and sensitivity. Furthermore, because the fluorescence intensity of reticulocytes is directly proportional to the erythrocyte RNA content, flow cytometric analysis using thiazole orange gives a quantitative reticulocyte maturity index (RMI). Previous studies have demonstrated that the RMI parameter is the earliest indicator of bone marrow engraftment following transplantation. In the present study, we analyzed the correlation of the RMI to standard red cell parameters, reticulocyte percentage, and absolute reticulocyte count in 413 anemic patients. The correlation of RMI to serum erythropoietin (Epo) and serum transferrin receptor (TfR) was analyzed in a subset of anemic blood samples. We found weak correlations between the RMI and hemoglobin (r2 = 0.041), hematocrit (r2 = 0.038), reticulocyte percentage (r2 = 0.078), and absolute reticulocyte count (r2 = 0.087). Stronger correlations were observed between the RMI and Epo (r2 = 0.181) and the TfR (r2 = 0.191). The results indicate that the RMI represents a cost‐effective measurement of erythropoietic activity and provides an additional parameter to classify anemic patients into categories of high and low erythropoietic activity, especially in hypoproductive anemias. © 1995 Wiley‐Liss, Inc.
Fluorescence drift detection as a novel QC procedure for DNA cell‐cycle analysis
Authors: Seamer Larry C. , Altobelli K. K. 
Published: June 2005

AbstractThe goal of this work was to develop an objective, quantitative, and reproducible method of detecting fluorescence drift which may have occurred during DNA cell‐cycle data acquisition. Quality control software, “TruPloid©” is described that analyzes list‐mode files to detect and quantify fluorescence drift using three separate statistical tests. We show that fluorescence drift may lead to a variety of measurement artifacts including high coefficients of variation, obscuring of small populations and creation of distinct artificial peaks. Forty percent of 50 archived list‐mode files displayed fluorescence drift, which demonstrates the need for detection methods to deal with this source of DNA cell‐cycle histogram artifacts. © 1995 Wiley‐Liss, Inc.
CD4 lymphocyte enumeration: Comparison between flow cytometry and enzyme immunoassay
Authors: Fei P. Cordiali , Solmone M. , Vanacore P. , Giglio A. , Bonifati C. , Carducci M. , Mussi A. , Chiarotti F. , Ameglio F. 
Published: June 2005

AbstractAssessment of the CD4 lymphocyte number, currently performed by flow cytometry, is one of the main laboratory tests for establishing progression to acquired immunodeficiency syndrome (AIDS). An enzyme immunoassay has recently been commercialized which can be very useful for counting CD4 cells in laboratories where flow cytometers are not available. In the present study, a comparative evaluation of CD4 positive lymphocytes with flow cytometry and the enzyme assay was made in healthy subjects (N = 30, R = 0.88, P < 0.0001), human immunodeficiency virus (HIV)‐infected individuals (N = 80, R = 0.91, P < 0.0001), and patients with autoimmune diseases (N = 28, R = 0.82, P < 0.001) or psoriasis (N = 18, R = 0.76, P = 0.01). A correlation between the two methodologies was not found in psoriatic patients under treatment with cyclosporin A (N = 7, R = 0.05, not significant). Some differences could be found at low CD4 lymphocyte levels since the influence of CD4 antigen eluted from monocytes or soluble CD4 in the whole blood sample could cause overestimation of CD4 cell numbers by the enzyme assay. © 1995 Wiley‐Liss, Inc.
Urinary sediment analyzed by flow cytometry
Authors: Yasui Yoshinori , Tatsumi Noriyuki , Park Keunsik , Koezuka Takuzo 
Published: June 2005

AbstractA flow cytometer for the automated analysis of urinary sediment was designed, and its performance was examined by the evaluation of 821 specimens. Auramine O, a day for DNA and RNA, was used for the staining of the sediment. Urine (5 ml or more) was processed by the instrument for sediment analysis. Conventional microscopic analysis was done for comparison. The RBC count, the WBC count, and the number of bacterial cells, epithelial cells, and casts found by the flow cytometer and by microscopy were compared. Correlation was high for all these results. The overall sensitivity, specificity, and efficiency (accuracy) in the items analyzed were 84.7%, 57.8%, and 67.2%, respectively. One hundred specimens could be analyzed by the instrument per hour. The instrument seemed useful for screening for urinary tract disorders to identify specimens that should be analyzed microscopically in routine laboratories. © 1995 Wiley‐Liss, Inc.
Prognostic significance of dna content in bladder cancer based on flow cytometric analysis of 249 transitional cell carcinomas
Authors: Vindeløv Lars L. , Christensen I. J. , Engelholm Svend A. , Guldhammer Birgitte H. , Højgaard Knud , Sørensen Bent L. , Wolf Hans 
Published: June 2005

AbstractThe prognostic significance of DNA index (DI), S‐phase fraction, and heterogeneity determined by flow cytometric DNA analysis was assessed in a prospective study of 249 newly diagnosed transitional cell carcinomas of the bladder. The median observation time was 4.8 years. A total of 456 subpopulations were detected. The S‐phases could be estimated in 299 subpopulations. A DI > 1.25 or an S‐phase above 9.7% were strongly correlated to invasiveness.One hundred and ten patients were treated with transurethral resection (TUR). Relapse‐free survival could not be predicted by the DNA‐derived parameters. Univariate analysis of survival showed prognostic significance of diploidy (0.98 < DI ≤ 1.02,p= 0.02), hypotetraploidy (1.50 < DI ≤ 1.96,P= 0.002), and S‐phase size (P= 0.008). Multivariate analysis pointed to the T‐classification (RR = 1.64) and hypotetraploidy (RR = 1.57) as prognostic parameters for survival of TUR‐treated patients.One hundred and thirty‐nine patients received radiotherapy (RT). A significantly better response was found for tumors with a subpopulation with a hypertetraploid DNA content (DI > 2.04,P= 0.05), and a significantly worse response for subpopulations with a maximum S‐phase > 24.5% (P= 0.04). T‐classification and histological grade had no predictive value. A logistic regression analysis indicated an estimated probability of response to RT of 77% for tumors with a DI > 2.04 and an S‐phase < 24.5%, whereas tumors with a DI < 2.04 and an S‐phase > 24.5% had only a 28% probability of response. The poor response to RT, predicted by an S‐phase > 24.5%, translated into a poor survival, whereas the better treatment response found for patients with a DI > 2.04 did not result in a longer survival. Multivariate analysis pointed to S‐phase (RR = 1.70), T‐classification (RR = 1.60), and grade (RR = 0.65) as independent prognostic parameters for survival of RT‐treated patients. © 1995 Wiley‐Liss, Inc.