bluuebunny/crossref_metadata_2025_split huggingface dataset:
Streptavidin‐based quantitative staining of intracellular antigens for flow cytometric analysis
Authors: Srivastava Praveer , Sladek Todd L. , Goodman M. Nadine , Jacobberger James W. 
Published: August 2005

AbstractA streptavidin–biotin‐based three‐step immunolabeling protocol for quantitative staining of intracellular antigens for flow cytometric analysis was evaluated using simian virus 40 (SV40) large T antigen. The concentration as well as the quantity of antibody used required optimization. The optimum labeling conditions varied moderately with cell lines that express T antigen levels over a 40–50‐fold range. The procedure resulted in specific fluorescence 2.4 times higher than that using a comparable two‐step indirect immunofluorescence technique. The gain in resolution was shown to be greater when staining cells with lower antigen levels. In the analysis of background fluorescence, the principal components were, as for the two‐step technique, autofluorescence and propidium spectral overlap. While streptavidin does add to the background, the increase is relatively small. Decreasing the propidium concentration from 50 μg/ml to 5 μg/ ml was found to reduce significantly the level of background from this source. Theoretical aspects of quantitative staiaing and of resolution versus quantification are discussed. © 1992 Wiley‐Liss, Inc.
Quantitation of resistance of leukemic cells to cytosine arabinoside from BrdUrd/DNA bivariate histograms
Authors: Lacombe F. , Belloc F. , Dumain P. , Puntous M. , Lopez F. , Bernard P. , Boisseau M. R. , Reiffers J. 
Published: August 2005

AbstractThe cytotoxicity of ara‐C derives from an inhibition of DNA synthesis after incorporation of ara‐CTP into DNA. The rate of DNA synthesis can be determined from the amount of bromodeoxy‐uridine (BrdUrd) incorporated into cells after a short exposure to BrdUrd. We developed a computer program to quantify the inhibition of the rate of DNA synthesis by analysis of the distribution of BrdUrd/ DNA. Inhibition was evaluated in ara‐C‐sensitive and resistant cells after incubation with different doses of ara‐C. An index of resistance to ara‐C (RI) was expressed as the ratio of the amount of BrdUrd incorporated into S phase cells incubated with ara‐C to that incorporated in the absence of ara‐C. In the ara‐C‐sensitive and resistant HL60 cells, a linear relationship between RI and log ara‐C concentration was observed. Small numbers of slightly resistant cells in mixtures of ara‐C‐sensitive and resistant cells could be determined using this method, making it suitable for clinical use to test the resistance of leukemic cells to ara‐C. © 1992 Wiley‐Liss, Inc.
Calcein: A novel marker for lymphocytes which enter lymph nodes
Authors: Weston Susan A. , Parish Christopher R. 
Published: August 2005

AbstractPrevious studies have identified unique cell surface antigens which are associated with the specific binding of lymphocytes to high endothelial venules (HEV). Evidence is presented in this paper which demonstrates that uptake of the fluorescent dye calcein by lymphocytes represents au additional marker for the lymph node homing subpopulation of lymphocytes. Calcein exhibits a characteristic ability to label lymphocytes differentially into two distinct populations, based on fluorescence intensity, that does not occur with three other structurally related, fluorescein‐based dyes. In vivo lymphocyte migration studies revealed that cells displaying the “dull” fluorescence phenotype, although entering all lymphoid organs examined, preferentially homed to the lymph nodes, particularly the popliteal lymph node (PLN). By contrast, lymphocytes displaying the “bright” phenotype were essentially excluded from entering lymphoid organs, where entry is HEV dependent, but were observed entering spleen, where entry is HEV independent. Furthermore, a high proportion (76. 5%) of lymphocytes displaying the dull fluorescence phenotype expressed the PLN homing receptor MEL‐14. Based on these observations it is suggested that calcein uptake may be a marker for general membrane properties, such as fluidity and plasticity, essential for the passage of lymphocytes through HEV. © 1992 Wiley‐Liss, Inc.
Automated image detection and segmentation in blood smears
Authors: Poon Steven S. S. , Ward Rabab K. , Palcic Branko 
Published: August 2005

AbstractA simple technique which automatically detects and then segments nucleated cells in Wright's giemsa‐stained blood smears is presented. Our method differs from others in (1) the simplicity of our algorithms; (2) inclusion of touching (as well as nontouching) cells; and (3) use of these algorithms to segment as well as to detect nucleated cells employing conventionally prepared smears. Our method involves: (1) acquisition of spectral images; (2) preprocessing the acquired images; (3) detection of single and touching cells in the scene; (4) segmentation of the cells into nuclear and cytoplasmic regions; and (5) postprocessing of the segmented regions. The first two steps of this algorithm are employed to obtain high‐quality images, to remove random noise, and to correct aberration and shading effects. Spectral information of the image is used in step 3 to segment the nucleated cells from the rest of the scene. Using the initial cell masks, nucleated cells which are just touching are detected and separated. Simple features are then extracted and conditions applied such that single nucleated cells are finally selected. In step 4, the intensity variations of the cells are then used to segment the nucleus from the cytoplasm. The success rate in segmenting the nucleated cells is between 81 and 93%. The major errors in segmentation of the nucleus and the cytoplasm in the recognized nucleated cells are 3.5% and 2.2%, respectively. © 1992 Wiley‐Liss, Inc.
Fluorescence ratio measurements of double‐labeled probes for multiple in situ hybridization by digital imaging microscopy
Authors: Nederlof P. M. , van der Flier S. , Vrolijk J. , Tanke H. J. , Raap A. K. 
Published: August 2005

AbstractTo expand the multiplicity of the in situ hybridization (ISH) procedure, which is presently limited by the number of fluorochromes spectrally separable in the microscope, a digital fluorescence ratio method is proposedFor this purpose, chromosome‐specific repetitive probes were double‐labeled with two haptens and hybridized to interphase nuclei of human peripheral blood lymphocytes. The haptens were immunocytochemically detected with specific antibodies conjugated with the fluorochromes FITC or TRITC. The FITC and TRITC fluorescence intensities of spots obtained with different doublehaptenized probes were measured, and the fluorescence ratio was calculated for each ISH spot. Combinations of different haptens, such as biotin, digoxigenin, fluorescein, sulfonate, acetyl amino fluorene (AAF), and mercury (Hg) were used. The fluorescence intensity ratio (FITC/ TRITC) of the ISH spots was fairly constant for all combinations used, with coefficients of variation between 10 and 30%To study the feasibility of a probe identification procedure on the basis of probe hapten ratios, one probe was double‐labeled with different ratios, by varying the relative concentrations of the modified nucleotides (biotin‐11‐dUTP and digoxigenin‐11‐dUTP) in the nick‐translation reactionMeasurement of the FITC and TRITC intensities of the ISH spots showed that the concentration of modified nucleotides used in the labeling procedures was reflected in the mean fluorescence intensity of the ISH spots. Furthermore, the ratio distributions showed little overlap due to the relatively small coefficients of variationThe results indicate that a multiple ISH procedure based on fluorescence ratio imaging of double‐labeled probes is feasible. © 1992 Wiley‐Liss, Inc.
Quantification of fluorescence in situ hybridization signals by image cytometry
Authors: Nederlof P. M. , van der Flier S. , Verwoerd N. P. , Vrolijk J. , Raap A. K. , Tanke H. J. 
Published: August 2005

AbstractIn this study we aimed at the development of a cytometric system for quantification of specific DNA sequences using fluorescence in situ hybridisation (ISH) and digital imaging microscopy. The cytochemical and cytometric aspects of a quantitative ISH procedure were investigated, using human peripheral blood lymphocyte interphase nuclei and probes detecting high copy number target sequences as a model system. These chromosome‐specific probes were labeled with biotin, digoxigenin, or fluorescein. The instrumentation requirements are evaluatedQuantification of the fluorescence ISH signals was performed using an epi‐fluorescence microscope with a multi‐wavelength illuminator, equipped with a cooled charge couple devices (CCD) camera. The performance of the system was evaluated using fluorescing beads and a homogeneously fluorescing specimenSpecific image analysis programs were developed for the automated segmentation and analysis of the images provided by ISH. Non‐uniform background fluorescence of the nuclei introduces problems in the image analysis segmentation procedures. Different procedures were tested. Up to 95% of the hybridization signals could be correctly segmented using digital filtering techniques (min‐max filter) to estimate local background intensitiesThe choice of the objective lens used for the collection of images was found to be extremely important. High magnification objectives with high numerical aperture, which are frequently used for visualization of fluorescence, are not optimal, since they do not have a sufficient depth of fieldThe system described was used for quantification of ISH signals and allowed accurate measurement of fluorescence spot intensities, as well as of fluorescence ratios obtained with double‐labeled probes. © 1992 Wiley‐Liss, Inc.
Immunoblotting technique to study release of melanophore‐stimulating hormone from individual melanotrope cells of the intermediate lobe of Xenopus laevis
Authors: de Rijk E. P. C. T. , Terlou M. , Cruijsen P. M. J. M. , Jenks B. G. , Roubos E. W. 
Published: August 2005

AbstractThe melanotrope cells in the pays intermedia in the pituitary of Xenopus laevis synthesize and release the melanophorestimulating hormone (αMSH), a small peptide that causes skin darkening during the process of background adaptation. Evidence has been found for a heterogeneity in biosynthetic activity of the melanotrope cells. In the present study two questions were addressed: (1) does the melanotrope cell population also show heterogeneous αMSH‐release, and (2) can this heterogeneity be changed by extracellular messengers? Since dopamine is known to inhibit αMSH‐release, this messenger is used to study the regulation of the heterogeneity. To quantify αMSH‐release from individual cells, a cell blotting procedure has been developed for the binding and relative quantification of the small αMSH peptide. The immunoblotting procedure involves binding of the cells to a carrier slide and binding of released αMSH to a nitrocellulose filter. After immunostaining, the amount of αMSH per cell was quantitated by image analysis. Untreated melanotrope cells reveal a distinct variability in αMSH‐release, some cells showing low secretory activity, whereas others are strongly secreting, indicating heterogeneity of αMSH‐release. Dopamine treatment strongly inhibits αMSH‐release from individual cells, resulting in a clearly less pronounced melanotrope cell heterogeneity. The effect of dopamine appears to be dose‐dependent as a low dopamine concentration has only a moderate effect on the αMSH‐release. It is proposed that dopamine is a physiological regulator of the degree of melanotrope cell heterogeneity in αMSH‐release. © 1992 Wiley‐Liss, Inc.
Comparison of BrdUrd and [3H]TdR incorporation to estimate cell proliferation, cell loss, and potential doubling time in tumor xenografts
Authors: Zätterström Ulf K. , Johansson Maria , Källén Anders , Baldetorp Bo , Oredsson Stina , Wennerberg Johan , Killander Dick 
Published: August 2005

AbstractIn this study, two different methods of estimating cell proliferation were compared: cell loss and potential growth rate of xenografted head and neck cancer grown in nude mice based on the detection of DNA incorporation of bromodeoxyuridine (BrdUrd) in one method, and [3H]thymidine ([3H]TdR) in the other. The 21‐d‐old xenografts were labelled in vivo, either with BrdUrd or [3H]TdR and excised at intervals during 65.5 h. In tumors containing BrdUrd, the percent labelling was measured in mid‐S and mid‐G1 phase windows of cytograms from bivariate DNA flow cytometry (FCM). In [3H] TdR‐labelled tumors, the percent labelled mitoses (PLM) was determined by light microscopy evaluation of autoradiographs. With a computer program based on a theoretical model, the percent labelling versus time after injection was used to analyze cell cycle time, cell loss tumor growth fraction, and potential doubling time. The values calculated from DNA incorporation with BrdUrd agreed well with those obtained from labelling with [3H]TdR, i.e., cell cycle time 2.3 vs. 2. 4 d, and growth fraction 67 vs. 70%. The estimated potential doubling time was 3.1 d and cell loss factor 40% by both methods. Flow cytometry analysis of BrdUrd‐labelling is considerably faster than the evaluation of [3H]TdR‐labelling, and the present results provide further support for the BrdUrd labelling method as a promising alternative to the PLM method in cell cycle studies designed to evaluate the relevance of cell proliferative properties in relation to biological behavior in xenografted head and neck cancer. © 1992 Wiley‐Liss, Inc.
Intercalation of anthracyclines into living cell DNA analyzed by flow cytometry
Authors: Belloc Francis , Lacombe Francis , Domain Patrice , Lopez Francis , Bernard Philippe , Boisseau Michel R. , Reifers Josy 
Published: August 2005

AbstractAnthracyclines (ANT) are used in the treatment of leukemia and other cancers. These drugs have beers shown to intercalate between the strands of DNA. In the present study, we show that the amount of ANT intercalated into DNA can be determined by measuring the fluorescence resonance energy transfer (FRET) between Hoechst 33342 (H33342) and ANT bound to DNA. The transfer efficiency was found to depend on the amount of disposable ANT but was independent of the amount of H33342 bound to DNA over a wide range of H33342 concentrations. The method was adapted for flow cytometric measurement of FRET in whole living cells and was used to evaluate the degree of intercalation of daunorubicin (DAU) and idarubicine (IDA) into DAU‐sensitive and DAU‐resistant leukemic cell lines. ANT intercalation into DNA was affected by factors which modify the intracytoplasmic concentration of ANT, and it was shown that the action of ANT and the resistance to ANT could not be attributed solely to the intercalative effect of the drugs. The method has advantages over previously described methods and represents a useful complementary tool in studies on the mode of action of ANT and the mechanisms of chemoresistance. © 1992 Wiley‐Liss, Inc.
Flow cytometric titration of retroviral expression vectors: Comparison of methods for analysis of immunofluorescence histograms derived from cells expressing low antigen levels
Authors: Sladek Todd L. , Jacobberger James W. 
Published: August 2005

AbstractFew quantitative studies addressing immunofluorescence histogram analysis have been published. One study by Overton (Cytometry 9:619–626, 1988) has shown threshold and histogram subtraction methods to be accurate for analysis of well‐separated immunofluorescence distributions of positive and negative cells. An evaluation of methods to analyze immunofluorescence histograms when positive and negative immunofluorescence distributions overlap has not, to our knowledge, been reported. In this paper, data obtained from flow cytometry of immunofluorescently stained cells infected with recombinant retroviruses that produce a range of simian virus 40 large T antigen levels were analyzed by threshold, histogram subtraction, and distribution modeling methods. This analysis showed that as the separation between the immunofluorescence distributions of positive and negative cell populations decrease the best methods for histogram analysis are modeling followed, in order, by histogram subtraction, and threshold analysis. © 1993 Wiley‐Liss, Inc.
Flow cytometric quantitation of nucleoside transporter sites on human leukemic cells
Authors: Jamieson Gary P. , Brocklebank Anne M. , Snook Marie B. , Sawyer William H. , Buolamwini John K. , Paterson Alan R. P. , Wiley James S. 
Published: August 2005

AbstractQuantitation of equilibrative, nitrobenzylthioinosine (NBMPR) sensitive (es) nucleoside transporters on blast cells isolated from patients with acute myeloblastic leukemia is useful in predicting intracellular accumulation of the antileukemic nucleoside drug, cytosine arabinoside. We previously reported the synthesis of a fluorescein‐labeled ligand for the es nucleoside transporter, 5‐(SAENTA‐x2)‐fluorescein. This paper reports the synthesis of 5‐(SAENTA‐x8)‐fluorescein in which the linkage between fluorescein and nucleoside ligand has been increased from 2 atoms to 8 atoms. This new ligand had a sixfold increase in affinity (Kd 0.9 ± 0.1 nM) as well as an 86% increase in the cell associated fluorescence output compared to its prototype 5‐(SAENTA‐x2)‐fluorescein. The fluorescence signal arising from 5‐(SAENTA‐x8)‐fluorescein specifically bound to freshly isolated and cultured leukemic myeloblasts was converted to molecules of equivalent soluble fluorescein (MESF) using standardized fluorescein microbeads and compared with the number of es nucleoside transporter sites assayed concurrently by [3H]NBMPR equilibrium binding analysis. A high correlation between the two assays was observed (r = 0.98), which enabled the cell‐bound fluorescence output of 5‐(SAENTA‐x8)‐fluorescein to be expressed in numbers of es nucleoside transporter sites per cell. The improved properties of 5‐(SAENTA‐x8)‐fluorescein over those of its prototype molecule make it a suitable reagent for flow cytometric quantitation of nucleoside transporter expression on leukemic cells isolated from patient samples. © 1993 Wiley‐Liss, Inc.
Increased sensitivity of damaged DNA to digestion with nuclease S1 as assessed in single cells by flow cytometry
Authors: Prosperi Ennio , Supino Rosanna , Bottiroli Giovanni 
Published: August 2005

AbstractDNA sensitivity to digestion with nuclease S1 was investigated in cells irradiated with gamma rays, or treated with the antitumor drug adriamycin (Adr). The nuclease‐resistant DNA fraction was determined by propidium iodide staining. Treated cells were found to be more sensitive to nuclease digestion than the undamaged controls. Gamma ray‐induced strand breaks were detectable at doses up to 10 Gy; an increase in the reaction temperature, from 37° to 63°C, was necessary in order to detect higher levels of damage. Nuclease S1 sensitivity in Adr‐treated cells showed a single‐peak, concentration‐dependent relationship, in agreement with the known self‐inhibitory effect exerted by high drug doses. Determination of DNA digestion could be performed in combination with other cellular parameters (e.g., protein content). Detection of drug‐resistant cells in a heterogeneous population of small‐cell lung carcinoma was achieved on the basis of the different sensitivity of the cells to enzymatic digestion. These results indicate that nuclease S1 may be a useful probe for studying in single cells DNA alterations induced by drugs or radiation. © 1993 Wiley‐Liss, Inc.
Rhodamine 123 fluorescence of immortal hybridoma cell lines as a function of glucose concentration
Authors: Borth Nicole , Kral Gerda , Katinger Hermann 
Published: August 2005

AbstractThe fluorescence of rhodamine 123 stained cells has been described to specifically reflect the activity of mitochondria. Changes in the intensity of fluorescence observed in stimulated lymphocytes were attributed to an increased glycolytic activity of cells due to increased growth rates. Previously reported changes in mitochondrial activity observed in batch cultures were likewise attributed to changed growth rates. In this study we report that the Rh123 fluorescence of hybridoma cell lines in batch culture more closely correlates to the glucose concentration in the culture supernatant than to growth rates. When cells are transferred into glutamine free medium with defined glucose concentrations ranging from 0 to 3,000 mg/L the mean Rh123 fluorescence adapts to the respective glucose concentration within 6 hours and gives a linear correlation. This can be explained by the previously described dependence of specific glucose consumption rates on glucose availability in the medium. The importance of controlling glucose availability, especially in large scale fermentations, is discussed. © 1993 Wiley‐Liss, Inc.
Neural network analysis of DNA flow cytometry histograms
Authors: Ravdin Peter M. , Clark Gary M. , Hough John J. , Owens Marilyn A. , McGuire William L. 
Published: August 2005

AbstractA pattern recognition system based on Neural Network Analysis, a form of artificial intelligence, was used to search DNA flow cytometry histograms for features that correlated with breast cancer patients' risk of relapse. DNA flow cytometry histograms and clinical followup information from 796 breast cancer patients were used to train a Neural Network to predict the clinical outcome of patients in a separate independent set of 794 patients. Median follow‐up in this patient data base was short, 23 months. Neural Network Analysis resulted in a model that evaluated DNA flow cytometry histograms differently than conventional analysis, which categorizes the histograms by ploidy and S‐phase fraction. Neural Network Analysis appeared to identify low risk and high risk subsets of patients as accurately as conventional analysis. Neural Network Analysis placed heavy emphasis on the region to the right of the diploid G2/M peak, where a subpopulation of nuclei with high DNA content is seen even in many histograms scored as diploid by conventional techniques. The number of nuclei in this region was found to be a powerful predictor of patient outcome, and multivariate analysis showed that the number of nuclei in this region and the S‐phase fraction both were independently predictive of relapse. This pilot study suggests that conventional analysis (based on a mechanistic interpretation of regions in flow cytometry histograms) might be used in conjunction with and improved by pattern recognition systems or insights derived from them. © 1993 Wiley‐Liss, Inc.