bluuebunny/crossref_metadata_2025_split huggingface dataset:
Cyanine dye labeling reagents containing isothiocyanate groups
Authors: Mujumdar Ratnakar B. , Ernst Lauren A. , Mujumdar Swati R. , Waggoner Alan S. 
Published: August 2005

AbstractNew isothiocyanate derivatives of cyanine dyes were synthesized as fluorescent covalent labeling reagents for proteins and other biomolecules. These dyes have maximum absorbance in the red and near infrared regions of the spectrum, have high extinction coefficients and have adequate quantum yields. Incorporating two alkyl sulfonate groups in the dye stuctures increases their water solubility, which is beneficial for labeling biological molecules in aqueous solution. Reactivities of proteins with these new cyanines are similar to their reactivities with fluorescein isothiocyanate. These new labeling reagents are complementary to the fluorescein and rhodamine reagents, expanding the possibilities of multicolor analyses. Sheep anti‐mouse‐IgG antibody was labeled with a pentamethine cyanine dye (CY5.8‐ITC) and used with a fluoresceinated antibody as a second reagent for detecting human T‐cell subsets by flow cytometry.
Three‐color fluorescence in situ hybridization for the simultaneous detection of multiple nucleic acid sequences
Authors: Nederlof P. M. , Robinson D. , Abuknesha R. , Wiegant J. , Hopman A. H. N. , Tanke H. J. , Raap A. K. 
Published: August 2005

AbstractA method is described for visualizing three nucleic acid sequences simultaneously by in situ hybridization using a new blue immunofluorescent label, amino methyl coumarin acetic acid (AMCA), in combination with green and red fluorescing FITC and TRITC.Three chromosome‐specific repetitive probes labeled with either amino acetyl fluorene (AAF), mercury, or biotin were hybridized simultaneously to metaphase chromosomes prepared from human blood lymphocytes or to interphase tumor nuclei. Conditions for the combined use of three immunocytochemical affinity systems as well as the optimal spectral separation of the three fluorescing labels have been determined.Three‐color in situ hybridization was applied to the study of numerical chromosome abnormalities as occur in human solid tumors. Further applications of this method in prenatal diagnosis for the detection of aneuploidy of the most frequently involved autosomes, as well as for the quantification of gene copy number and mRNA expression, are discussed.
Automatic analysis of flow cytometric DNA histograms from irradiated mouse male germ cells
Authors: Lampariello Francesco , Mauro Francesco , And Raffaella Uccelli , Spanò Marcello 
Published: August 2005

AbstractAn automatic procedure for recovering the DNA content distribution of mouse irradiated testis cells from flow cytometric histograms is presented. First, a suitable mathematical model is developed, to represent the pattern of DNA content and fluorescence distribution in the sample. Then a parameter estimation procedure, based on the maximum likelihood approach, is constructed by means of an optimization technique. This procedure has been applied to a set of DNA histograms relative to different doses of 0.4‐MeV neutrons and to different time intervals after irradiation. In each case, a good agreement between the measured histograms and the corresponding fits has been obtained. The results indicate that the proposed method for the quantitative analysis of germ cell DNA histograms can be usefully applied to the study of the cytotoxic and mutagenic action of agents of toxicological interest such as ionizing radiations.
A simple preservative for flow cytometric DNA analysis
Authors: Alanen Kalle A. , Klemi Pekka J. , Taimela Simo , Joensuu Heikki 
Published: August 2005

AbstractA solution containing citric acid buffered saline (CABS) and 99% ethanol (E) 1:1 was used for preserving cells for flow cytometric DNA analysis. DNA histograms obtained from fine needle biopsy aspirates and preserved in CABS+E had a similar mean coefficient of variation (CV) as was obtained from aspirates taken in CABS (3.3 vs. 3.4%) and a clearly smaller mean CV than was obtained from aspirates preserved in 50% ethanol (mean 4.8%, P < .0001). Aspirates taken in CABS more often contained a small (< 3,000) number of cells as compared with aspirates preserved either in CABS+E or ethanol (P < .0001). Since preservation of cells in CABS+E allows long‐term storage of samples and results in a decreased number of insufficient samples as compared with buffered saline and in an enhanced resolution as compared with 50% ethanol, CABS+E is recommended for preservation of cytological samples to be analyzed for DNA content with flow cytometry.
Modification of a trypsin‐detergent method for DNA flow cytometry of human epidermis
Authors: Stough Dowling B. , Burns E. Robert , Mallory Susan B. , Pipkin James L. , Hinson William G. 
Published: August 2005

AbstractAn existing technique (Vindelov et al. Cytometry 3:323, 1983) has been modified for DNA flow cytometry of human epidermis obtained from 2 to 3 mm punch biopsies. By varying the length of time of digestion of the epidermal disc by trypsin from 5 to 70 min a controlled release of keratinocytes occurred beginning with the stratum basale and proceeding toward, but not including, the superficial layer of the epidermis, the stratum corneum.
Efficient use of monoclonal antibodies for immunofluorescence
Authors: Edwards Bruce S. , Shopp George M. 
Published: August 2005

AbstractWe describe here a simple and rapid small volume microplate‐based immunofluorescence staining method in which fluorochrome‐conjugated monoclonal antibodies (MAb) from three different manufacturers, used at a single standardized quantity (50 ng per test), resulted in optimal staining of human lymphocyte subsets. Staining reactions were robust, in that the number of lymphocytes used could be varied over a wide range (3 × 104‐1 × l06 cells per microplate well) without significant effects on the fluorescence intensity of staining or nonspecific binding by MAb. A measure of the efficiency of MAb use was the number of tests theoretically possible to perform with nominal 100 test kits; this figure ranged from 400 to 20,000 tests, depending on the MAb in question. This method was readily adaptable to both single‐and two‐color immunofluorescence analysis.
Detection by flow cytometry of protoplast fusion and transient expression of transferred heterologous CD4 sequences in COS‐7 cells
Authors: Rice Glenn C. , Pennica Diane 
Published: August 2005

AbstractTransfer and expression of a plasmid containing the gene encoding the human T‐cell antigen CD4 by protoplast fusion was measured by flow cytometry (FCM). Protoplasts were prelabeled with fluorescein isothiocyanate (FITC) and fused to COS‐7 cells. Nonspecific protoplast adsorption to the plasma membrane was differentiated from successful protoplast fusion by the addition of an antibody directed against fluorescein to quench extracellular protoplast fluorescence. Transfection efficiencies were defined as both percent CD4 expressing cells and CD4 expression levels on a single cell basis in the transient immunofluorescence assay. Cell sorting studies indicated that intracellular protoplast‐associated fluorescence immediately after fusion exhibited a good correlation with transient CD4 transfection efficiencies as measured by indirect immunofluorescence. Reconstruction experiments comparing CD4 transfer efficiencies of protoplast fusion and calcium phosphate transfection showed that fusion resulted in a higher percentage of CD4 expressing tranfectants, while calcium phosphate transfection yielded higher CD4 expression levels on a single cell basis. Thus, FCM appears to be useful as a new tool for sensitive detection of transient expression of heterologous reporter genes in COS‐7 cell.
Chromosome banding analysis by slit‐scan flow cytometry
Authors: Bartholdi Marty F. , Meyne Julie , Johnston Roger G. , Cram L. Scott 
Published: August 2005

AbstractWe have investigated the use of fluorescence banding patterns for the resolution of metaphase chromosomes by slit‐scan flow cytometry. Fluorescence scans of R‐banded chromosomes have been obtained for the entire human karyotype. Metaphase chromosomes were R‐banded in suspension by staining with chromomycin A3 after hypotonic treatment in Ohnuki's buffer. Specific fluorescent landmark bands were detected for human chromosomes 1–12. Scans obtained for chromosomes 13–22 did not contain sufficient information for classification. Characteristic fluorescence patterns for human chromosomes 1 and 3 provided the clearest evidence for the detection of R‐bands by slit‐scan flow cytometry. Specific patterns were detected for human chromosomes 9–12 in which the number and placement of the fluorescent bands served as classifiers.
Vita blue: A new 633‐nm excitable fluorescent dye for cell analysis
Authors: Lee Linda G. , Berry Gillian M. , Chen Chia‐Huei 
Published: August 2005

AbstractSeveral new derivatives of fluorescein were synthesized. The dyes were characterized by NMR; and the absorbance, excitation, and emission spectra were measured. The fluorescence quantum yields of the dyes were determinde. The pKa3 values of the dyes were measured by fluorescence titration. The characteristics of the fluorescein and sulfonefluorescein derivatives were compared. The most promising dye for use in cell analysis appeared to be compound 9, which was given the name Vita Blue. The dibutyrate ester of Vita Blue was made and the compound was given the name Vita Blue dibutyrate (VBDB, 14). The Km of VBDB with pig liver esterase was measured and found to be 4 × 10−5 M. The pKa3 of Vita Blue was 7.56 ± 0.03; both acidic and basic forms were fluorescent (dual fluorescence). The use of VBDB as an intramolecular esterase substrate and its utility for the discrimination between live and dead cells by flow cytometry is described.
Fluorescein isothiocyanate staining intensity as a probe of hyperthermia‐induced changes in chromatin conformation
Authors: Dyson J. E. D. , Britten R. A. , Battersby I. , Surrey C. R. 
Published: August 2005

AbstractIn a previous report we presented evidence for large increases in fluorescein isothiocyanate (FITC) fluorescent intensity caused by hyperthermia which were not associated with synthesis of heat‐shock proteins. We have now refined and considerably extended the measurements of increases in FITC fluorescent intensity caused by hyperthermia within the range 41.0°C to 50.0deg;C, and associated these with the extent of cell death caused by the hyperthermia. It appears that cell death ensues when the FITC fluorescent intensity has not returned to its baseline value within the time of one cell cycle. If thermotolerance is induced, there is a concomitant reduction in the increase in FITC staining intensity and the extent of cell death. When hyperthermia is followed by acid extraction, an additional increase in FITC staining intensity (above that due to hyperthermia alone) is observed, indicating separate sites of action on basic nuclear proteins. Hyperthermia and acid extraction have related effects on the relationship between FITC and propidium iodide staining. Hyperthermia‐induced increases in FITC staining intensity are almost completely reversed by 6.7 mM formaldehyde with a marginal effect on the control FITC staining at this formaldehyde concentration. We suggest that hyperthermia causes extensive dissociation of basic protein‐protein binding within nuclear chromatin, and that this may be a contributory cause of hyperthermia‐in‐duced cell death.
Flow cytometric studies of the nuclear matrix
Authors: Wright William D. , Higashikubo Ryuji , Roti Roti Joseph L. 
Published: August 2005

AbstractWe have devised a method to measure the protein and nucleic acid content of the nuclear matrix using flow cytometry. Nuclear matrices were prepared from nuclei by DNase I digestion followed by 3 M NaCl extraction. The resulting particles were stained with fluorescein isothiocyanate (FITC) for protein and propidium iodide (PI) for double‐stranded nucleic acids, and fluorescence as well as forward angle light scatter was detected. The matrices were also subjected to additional chemical or enzymatic perturbations, and changes in the above parameters were measured. Results showed that matrices from heat‐shocked cells not onlyretained the majority of heat‐induced excess nuclear protein, but also exhibited higher PI signals than controls after RNase A digestion. This observation did not hold if RNase A digestion preceded high‐salt extraction, suggesting that a salt‐extractable moiety had been replaced or altered by heat so that double‐stranded RNA was protected from the nucleolytic attack. The residual PI fluorescence in matrices from heated cells bore a linear relationship to the increased protein content in those matrices, indicating that the excess protein sequesters matrix‐associated RNA. Polyacrylamide gel electrophoresis of matrix polypeptides revealed increased amounts of many proteins as a result of heat as well as the appearance of several new proteins, one of which comigrates with the HSP72/73 heat‐shock proteins. The results of these studies show that flow cytometry can be used to study the nuclear matrix and is capable of detecting changes that result from alterations in its protein composition.
Intratumoral variations in dna distribution patterns in mammary adenocarcinomas
Authors: Askensten Ursula G. , Von Rosen Anette K. , Nilsson Roland S. , Auer Gert U. 
Published: August 2005

AbstractCorrelated flow‐cytometric (FCM) and microspectrophotometric (MSP) techniques were applied to investigating whether intratumoral variations in the DNA distribution patterns of 21 primary mammary adenocarcinomas can occur. Although neoplastic cell populations with both diploid and tetraploid (i.e., euploid) distribution patterns could be found in varying proportions in some of the tumors, there was no evidence in any tumor nodule for the presence of euploid populations in one part and aneuploid populations in another. This statement was based on the results of the MSP technique, where the assessments were made on cytodiagnostically identified neoplastic cells. Also, when applying the FCM technique the statement was found to be essentially valid; only one of the tumor nodules showed a DNA distribution pattern that, by means of the criteria used in this procedure, was defined as being both euploid and aneuploid. Here, however, the technique consists of assessments made on a great number of microscopically non‐identified cells.It was concluded that when conflicting reports are given from different laboratories on the prognostic value of the cytochemically assessed DNA distribution patterns in breast carcinomas, they are not likely to be attributed to intratumoral DNA heterogeneity but, rather, to differences in the methods used and in the criteria applied for the so‐called ploidy assessments.
Initial evaluation of a new epithelial antigen (T16) for bivariate flow cytometry of bladder irrigation specimens
Authors: Bretton P. R. , Myc A. , Cordon‐Cardo C. , DeAngelis P. , Fair W. R. , Melamed M. R. 
Published: August 2005

AbstractBivariate flow cytometry (FCM) was used to study immunofluorescent T16 mouse monoclonal antibody (Mab) binding simultaneously with propidium iodide DNA measurements in bladder irrigation specimens from 30 patients with a history of bladder cancer. Aliquots of the same samples were stained with acridine orange (AO) and examined by conventional FCM. T16 Mab is believed to be specific for epithelial cells in this type of specimen and stained from 13% of the cells in a patient with cystitis to 95% of the cells in a patient with an atypical papilloma. In combination with DNA measurements, this antibody increased the sensitivity of FCM in patients with severe cystitis and relatively small numbers of tumor cells, but the diagnostic specificity may be decreased and the criteria established for interpreting univariate flow cytometry may have to be re‐evaluated and modified.
DNA flow cytometry in sperm cells from unilaterally orchiectomized patients with testicular cancer before further treatment
Authors: Fosså Sophie D. , Melvik Jan E. , Juul Niels O. , Pettersen Erik O. , Theodorsen Liv 
Published: August 2005

AbstractLight microscopic sperm cell analysis and DNA flow cytometry in the seminal fluid were done in 85 testicular cancer patients after orchiectomy before further treatment. The results were compared with those from 26 healthy age‐matched males (control group). A computer‐based method for analysis of the DNA histograms was developed for evaluation of the percentage of sperm cells within the sub‐haploid, haploid (1c), and diploid (2c) and > 2c levels. Compared with the control group, testicular cancer patients had a reduced sperm cell density and sperm cell motility. The mobility grade was also significantly reduced as compared with healthy males. In addition, the number of condensed haploid sperm cells (within the subhaploid level) was decreased in testicular cancer patients, whereas the percentages of noncondensed haploid (1c), diploid, and > 2c cells were increased. Most of the DNA flow cytometric parameters were significantly correlated with sperm cell density. DNA flow cytometry in human seminal fluid offers a possible means of assessing spermatogenesis, thus providing an objective method for studying fertility disturbances, for example, in cancer patients before and after treatment.
Differences in the flow and absorption cytometric DNA distributions of mouse hepatocytes and tumor cells
Authors: Bose Kallol K. , Curley Steven , Smith William J. , Allison David C. 
Published: August 2005

AbstractWe have determined the DNA content, the ploidy levels, and the percentages of different cell types present in small and large mouse mammary tumors as well as in young and old mouse livers by using absorption and flow cytometry. Absorption cytometry data indicated a significant increase in the proportion of transformed G0/G1 cells in the tumors as compared to that of the stromal G0/G1 cells with progressive tumor growth. This increase was not detected by flow cytometry. In both young and old mouse livers, a small number of cells of higher ploidy (8C and 16C) were detected by absorption cytometry but were not apparent in histograms obatined by flow cytometry. Furthermore, changes in the proportions of liver cells of different ploidy with age were apparent in absorption cytometry data but not in flow cytometry data. In one mouse liver experiment, a 6C cell peak appeared in the flow cytometry histogram, but a direct measurement of DNA content by absorption cytometry failed to detect cells with such a peak. We therefore believe that some caution may be warranted in the use of flow cytometry alone for evaluation of DNA distributions and of the proportions of different types of cells in complex solid tissues.
Problem of size dependence in fluorescence DNA cytometry
Authors: Hamada Shinshichi , Fujita Setsuya 
Published: August 2005

AbstractThe relationship among fluorescence intensity, area, thickness, and DNA concentration of DNA‐bearing polyacrylamide film, stained with 50 ng/ml DAPI, was studied using an epi‐illumination cytofluorometer. Measurements and theoretical analysis suggest the inevitability of the size‐dependent effect on human cell nuclei in which the order of the DNA concentration is approximately 10 mg/ml, and that this effect can be virtually negated if the nuclei are stretched and flattened to less than 0.33 μm in thickness on the smears. Alternative practical ways to minimize this effect are to decrease the dye concentration of a staining solution and to make smears as thin and uniform as possible by some means such as hypotonic treatment and/or using an automatic centrifuge smearing apparatus. The size‐dependent effect, nevertheless, should be taken into account particularly when determining the DNA content of different cell types and the DNA aneuploidy of tumor cells, regardless of the technique used for measurement.
Measurement of S‐phase fractions in lymphoid tissue comparing fresh versus paraffin‐embedded tissue and 4′,6′ ‐diamidino‐2 phenylindole dihydrochloride versus propidium iodide staining
Authors: Camplejohn R. S. , Macartney J. C. , Morris R. W. 
Published: August 2005

AbstractS‐phase fractions for 62 lymphoid biopsies were calculated, by means of flow cytometry, from both fresh and paraffin‐embedded tissue. The purposes of this study were to determine whether significant differences were seen between S‐phase estimates obtained from fresh and fixed tissue and to compare results obtained with two DNA dyes, namely 4′‐6′‐diamidino‐2 phenylindole dihydrochloride (DAPI) and propidium iodide (PI). The 62 cases consisted of 38 cases of non‐Hodgkin's lymphoma (NHL), 19 reactive samples, and 5 cases of Hodgkin's disease. Fifty‐four of the samples showed DNA diploid profiles. A good agreement between S‐phase results from fresh and fixed tissue was seen, with technical factors accounting for around 20% of the total variance. Using a paired t test, no significant difference was seen between fresh and fixed tissue for diploid cases, but there was a trend for S‐phase estimates from fixed tissue to be higher. If all cases (including the eight DNA aneuploid samples) were included in the analysis this difference just reached statistical significance (p <.05). In a subgroup of 19 of the cases, a comparison was performed on both fresh and fixed tissue of staining with DAPI and PI. A good agreement between results with both DNA stains was found on fresh and fixed tissue, with no significant differences being apparent, and stain‐related factors accounted for only 10% of the total variance.
Comprehensive quality assessment approach for flow cytometric immunophenotyping of human lymphocytes
Authors: Edwards Bruce S. , Altobelli Kathleen K. , Nolla Hector A. , Harper Donna A. , Hoffman Robyn R. 
Published: August 2005

AbstractFlow cytometric immunophenotypic (IPT) evaluation has become an important adjunct to clinical patient management and epidemiological studies. This has precipitated a need for stringent quality assessment (QA) procedures to ascertain data integrity. We evaluated a QA approach to monitor all elements of the immunophenotyping process, inclusive of blood collection and processing procedures as well as of staining reagent and instrument performance. Central to our approach was preparation each day, in parallel with clinical analytes, of lymphocytes from healthy donors, selected from a 15 donor panel. IPT parameters evaluated over a 19 month period included frequencies of CD3+, CD4+, CD8+, and CD20+ lymphocytes and the ratio of CD4+ to CD8+ lymphocytes. The sensitivity for analytical error detection was reflected by median coefficients of variation of these parameters within individual panel donors, which were 4.1%, 4.5%, 3.9%, 8.2%, and 10.1%, respectively. IPT parameter values were determined each day for two of the panel donors, then averaged and standardized to obtain a quality or Q variate, which was the basis of QA. Error detection sensitivity decreased 0.6–1.7% and the number of false rejections increased 1.2–3.3% when one panel donor rather than two was used daily for QA. This study also illuminated important aspects of what constitutes the norm for longitudinal IPT parameter variation in healthy individuals including: (1) a generally low degree of temporal parameter variation within individual donors, but (2) significant differences between donors with respect to variance estimates for CD3+ and CD8+ lymphocyte frequencies and CD4+/CD8+ lymphocyte ratios, and (3) an apparent seasonal pattern of variation in CD4+ T‐cell frequencies.