bluuebunny/crossref_metadata_2025_split huggingface dataset:
Use of the biotinylated antibody DAKO‐ER 1D5 to measure oestrogen receptor on cytokeratin positive cells obtained from primary breast cancer cells
Authors: Brotherick I. , Lennard T. W. J. , Cook S. , Johnstone R. , Angus B. , Winthereik M. P. , Shenton B. K. 
Published: June 2005

AbstractA method for the use of a biotinylated antibody (DAKO‐ER 1D5) to quantify oestrogen receptors (ER) on tumour cells by flow cytometry is described. ER quantification was determined after treatment with saponin rendering cells permeable to ER antibody. Use of dual parameter labelling was performed utilizing a FITC‐conjugated antibody (NCL‐5D3) directed against cytokeratin 8/18. This allowed selection of breast cancer cells of epithelial origin by gating to exclude contaminating inflammatory and stromal cells. Use of such a gating technique was seen to identify cells with a higher level of ER expression. Using QC quantum bead standards, the number of ER binding sites per cell was assessed. Results were compared with conventional ER quantification using a radio‐ligand binding assay. A high degree of correlation was found between the two methods. The flow cytometric method for ER quantification described is simple, rapid, and reproducible. The assay may be of particular value in measuring ER on urgent clinical samples. Advantages of this assay over the radio‐ligand binding assay include reduction in use of radio‐labelled iodine compounds, a decrease in analysis time, and reduced cost and quantity of material needed for assay. © 1995 Wiley‐Liss, Inc.
Quantitative cytofluorimetric determination of cell membrane‐associated large tumor antigen on SV40‐transformed cells
Authors: Hess Ralf D. , Kuther Markus , Haessler Christel , Paetzold Susanne , Braun Dietmar G. , Brandner Gerhard 
Published: June 2005

AbstractThe aim of this study was to quantitate the number of cell membrane‐located SV40 large tumor antigen (large T) molecules of SV40‐transformed cell lines by cytofluorimetric analysis. Five different SV40‐transformed cell lines were labelled by either a biotin‐ or a fluorescein‐conjugated monoclonal antibody, PAb1605, which is specific for the large T carboxyterminus. The conjugated‐antibody fluorescence signals of the stained large T molecules of transformed cells were measured via cytotluorimetry. Comparison of the fluorescence signals of calibrated beads bearing a known number of fluorescein molecules to the signals of conjugated PAb1605 antibodies bound on microbeads to a defined number of IgG binding sites made it possible to determine the number of antibody‐accessible large T molecules per SV40‐transformed cell. The numbers (×10−4) found per cell were 1.0 (ELONA, hamster), 3.0 (VLM, mouse), 3.5 (mKSA, mouse), 11 (C57SV, mouse), and 5.5 (SV80, human), respectively. Thus, the technique described allows a precise quantitation of surfaceexposed, antibody‐accessible viral antigen expression. © 1995 Wiley‐Liss, Inc.
Scanning microfluorometric analysis of proliferating cell nuclear antigen in formalin‐fixed sections of hyperplastic and neoplastic rat liver
Authors: Kong Jin , Ringer David P. 
Published: June 2005

AbstractScanning laser cytometric analysis of fluorochrome‐labeled cells was used to survey and quantitate the distribution of proliferating cells in formalin‐fixed, paraffin‐embedded tissue sections from hyperplastic and neoplastic rat livers. The technique used fluorescent immunochemical staining of proliferating cell nuclear antigen (PCNA) as a marker for proliferating cells and propidium iodide as a fluorescent nuclear counterstain. Use of an antigen retrieval treatment improved detection of PCNA and treatment of tissue sections with crystal violet improved the sensitivity of the method by quenching background autofluorescence. PCNA evaluation of cell proliferation in regenerating rat liver 0–48 h post‐partial hepatectomy showed that 3–43% of cells stained positively for PCNA, a pattern closely correlating with previously reported rates of maximum DNA synthesis. The PCNA staining patterns observed among cells in neoplastic nodules were more focal in distribution and indicated that from 5 to 25% of the cell nuclei per nodular region stained positively for PCNA. This use of image analysis for the rapid identification of proliferating cell areas in fixed, paraffin‐embedded tissue active in neoplastic growth will expedite in situ cytochemical and molecular studies attempting to identify key differences between hyperplastic and neoplastic growth. © 1995 Wiley‐Liss, Inc.
Relationship between DNA ploidy level and tumor sociology behavior in 12 nervous cell lines
Authors: Kiss Robert , Camby Isabelle , Salmon Isabelle , Van Ham Philippe , Brotchi Jacques , Pasteels Jean‐Lambert 
Published: June 2005

AbstractCell population sociology was studied in two medulloblastomas and 10 astrocytic human tumor cell lines by means of the characterization of the structure of neoplastic cell colonies growing on histological slides. This was carried out via digital cell image analysis of Feulgen‐stained nuclei, to which the Delaunay triangulation and Voronoi paving mathematical techniques were applied. Such assessments were compared to the DNA ploidy level (assessed by means of DNA histogram typing). The results show that the cell colony architecture characteristics differed markedly according to whether the cell lines were euploid (diploid or tetraploid) or aneuploid (hyperdiploid, triploid, hypertriploid, or polymorphic). In fact, the cell colonies from the euploid cell nuclei populations were larger and more dense than those from the aneuploid ones. Furthermore, for an identical period of culture, the cell lines from high‐grade malignant astrocytic tumors (glioblastomas) exhibited cell colonies that were larger and more dense than those in cell lines from low‐grade astrocytic tumors (astrocytomas). In each of these two groups, the diploid cell nuclei populations exhibited cell colonies larger and more dense than the nondiploid colonies. The present methodology is now being applied in vivo to histological sections of surgically removed human brain tumors in order to distinguish between high‐risk clinical subgroups and medium‐risk subgroups in clearly circumscribed histopathological groups. © 1995 Wiley‐Liss, Inc.
Flow cytometric detection of glutathione S‐transferase isoenzymes by quantitative immunofluorescence under nonsaturating conditions
Authors: Pifferi Antonio , Filippini Cristian , Capolongo Laura , Codegoni Annamaria , Balconi Giovanna , Ubezio Paolo , Tagliabue Giovanna 
Published: June 2005

AbstractThe glutathione (GSH)‐glutathione S‐transferase (GST) detoxification system is an important element in cellular defence against injurious agents and anticancer drugs. GST isoenzymes may represent biochemical markers of neoplastic transformation, and, possibly, drug resistance is associated with altered GST‐isoenzyme levels. The ability to measure GST‐isoenzymes in cell populations would be useful for several biological and clinical applications. We have developed an immunofluorescence flow cytometric method for the simultaneous detection of different GST‐isoenzymes and of DNA in fixed cells. Due to the impossibility of working under saturating conditions for the anti‐GST antibody, a normalizing procedure was developed to permit quantitative analysis of single cells labelled with the anti‐GST antibody at high dilution. A theoretical model and experimental data supported the use of this procedure. The method proposed is general and could be applied to other antibodies in order to obtain quantitative data outside saturating conditions. The method was challenged in different applications in order to compare it with other classical techniques. First, we characterized sublines resistant to different anticancer drugs with respect to variations of GST isotypes. In a second application, we studied the intercellular heterogeneity of GST content in mouse renal cells. In addition, GST was determined in aneuploid cells from solid tumor biopsies by separation from diploid cells on the basis of DNA content. Finally, GST distribution during cell‐cycle progression was studied in two different cell lines by the biparametric analysis of GST/DNA. © 1995 Wiley‐Liss, Inc.
Measurement of tumor necrosis factor activity by flow cytometry
Authors: Lévesque Ann , Paquet Alain , Pagé Michel 
Published: June 2005

AbstractTumor necrosis factor‐α (TNF‐α) is a monokine of 17 kDa produced by activated macrophages and various cells involved in the immune system. We propose a new method for the measurement of TNF activity using flow cytometry. After an incubation with TNF, L929 cells were harvested and treated with a calcein‐AM and ethidium homodimer‐1 solution. Nonfluorescent calcein‐AM is hydrolyzed by intracellular esterases to yield fluorescent calcein. The ethidium homodimer‐1 is a high‐affinity red fluorescent DNA dye that is internalized only through altered cell membranes. A very good correlation was observed between the calcein fluorescence intensity and the number of viable cells as well as the ethidium fluorescence and the number of cells with altered membranes. The assay is sensitive, inexpensive, and correlates with the already reported crystal violet assay while measuring membrane alteration by TNF. It allows the simultaneous measurement of total living and dead cells. There is no interference with culture medium components. This method is rapid and may be used for routine measurement of TNF activity. © 1995 Wiley‐Liss, Inc.
Use of a spherical multiparameter transducer for flow cytometry
Authors: Leif R. C. , Cayer M. L. , Dailey W. , Stribling T. , Gordon K. 
Published: June 2005

AbstractFused silicon dioxide, multiparameter flow transducers with 50 μm internal square cross section and ∼60 μm length can simultaneously measure DC and RF impedance as well as fluorescence and multiple‐angle light scattering. A spherical version of such a transducer was mounted in an EPICS CVA flow‐cell housing and was installed on a research prototype equipped with an argon‐ion laser. The signal that was produced by the spherical transducer with EPICS DNA‐Check beads was 1.73 times greater than that produced with the standard cylindrical flow cell. Similarly, with EPICS Immuno‐Brite beads, the average ratio was 1.96.The Coulter impedance and light‐scattering measurements were similar to those produced with the conventional cylindrical outside flow cell, although the internal cross section of the sphere was square and that of the cylinder was circular. The theoretical arguments of Leif and Wells have been demonstrated to be correct. At present, monolithic, spherical fused‐silica transducers are the optimal design for combined electrooptical, multiparameter flow cytometry analyzers. © 1995 Wiley‐Liss, Inc.
Methods for cell proliferation analysis by fluorescent image cytometry
Authors: Souchier Catherine , Ffrench Martine , Benchaib Mehdi , Catallo Regine , Bryon Paul Andre 
Published: June 2005

AbstractMethods were developed for multimodal microscopic image analysis in order to identify and analyze one cell type under various microscopic conditions. Our purpose was to quantify both total DNA content using propidium iodide (PI) stain and S‐phase fraction using the bromodeoxyuridine (BrdUrd) incorporation technique in cell population subsets. The model chosen was plasma cells in bone marrow triply labelled with fluorescein isothiocyanate (FITC) for intracytoplasmic immunoglobulins, with amino‐methylcoumarin‐acetate (AMCA) for BrdUrd, and with PI for DNA. Image analysis included three phases. First, plasma cells were recognized on FITC images, and the centroid positions were stored. Second, plasma cell nuclei were geodesically reconstructed from these stored positions using PI images in which DNA content was measured, and the nuclear mask outlines were stored. Third, BrdUrd incorporation level of plasma cells was measured on AMCA images inside PI nuclei masks and stored. Image DNA vs. BrdUrd scatterplots were obtained for cells selected according to the expression of intracytoplasmic immunoglobulin. Thus, both ploidy and proliferation could be independently evaluated on a subset of the cellular population. © 1995 Wiley‐Liss, Inc.
Further characterisation of the in situ terminal deoxynucleotidyl transferase (TdT) assay for the flow cytometric analysis of apoptosis in drug resistant and drug sensitive leukaemic cells
Authors: Chapman Rachel S. , Chresta Christine M. , Herberg Ali Alejo , Beere Helen M. , Heer Sukhbindar , Whetton Anthony D. , Hickman John A. , Dive Caroline 
Published: June 2005

AbstractApoptosis, originally defined by specific morphological changes, is characterised biochemically by non‐random cleavage of DNA. Depending on cell type, this DNA cleavage proceeds from 300 and 50kbp fragments prior to, concomitantly with, or in the absence of 180bp integer fragmentation. Incorporation into fragmented DNA of biotin‐labelled nucleotides by terminal deoxynucleotidyl transferase (TdT) has recently become a standard flow cytometric assay for the identification and quantitation of apoptosis. Nucleotide incorportion is visualized using avidin‐tagged fluorescein isothiocyanate (FITC) (Gorczyca et al.: Cancer Res 53:1945–1951, 1993; Jonker et al.: Cytometry (Suppl 13):Abstr 99A, 1993). Here, we characterise this assay further in three different haemopoietic cell lines. Drug‐induced DNA damage is not identified by the TdT assay unless it is coupled to the apoptotic response. This was demonstrated using cells in which activation of the oncogenic Abelson‐encoded protein tyrosine kinase suppressed drug‐induced apoptosis, but did not inhibit drug‐induced DNA damage (by melphalan, hydroxyurea, or etoposide). Furthermore, the TdT assay identifies DNA fragments formed during apoptosis induced by etoposide and N‐methylformamide in HL60 and MOLT‐4 cells, including those high molecular weight DNA fragments formed in MOLT‐4 cells which were not further cleaved to 180–200bp integer fragments. Our results support the use of flow cytometry and the TdT assay to reliably measure apoptotic cells in heterogeneous cell samples. © 1995 Wiley‐Liss, Inc.
Estimation of fetal hemoglobin levels in individual red cells via fluorescence image cytometry
Authors: Horiuchi Kazumi , Osterhout Melissa L. , Kamma Hiroshi , Bekoe Nicoletta A. , Hirokawa Kevin J. 
Published: June 2005

AbstractA method for estimating fetal hemoglobin (Hb F) levels in individual red blood cells was developed. Cell smears were prepared using a slide maker to ensure uniform thickness and were then stained with immunofluorescence. An antifading gel was applied to preserve a stable fluorescence. The total fluorescence intensities from the same number of red cells in different slide specimens correlated with their hemolysate Hb F levels, which were determined via column chromatography (R = 0.95). Hb F level in individual cells was estimated from fluorescence intensity and cell area, which were determined via image analysis techniques and the hemolysate Hb F level. Blood from a normal subject, a subject with hereditary persistence of fetal hemoglobin, and from sickle cell patients with varying Hb F levels was analyzed. Our analyses showed a wide distribution of Hb F among cells for the normal subject and a gaussian distribution with a peak at the hemolysate Hb F level for the subject with hereditary persistence of fetal hemoglobin. The Hb F distributions were unique to the patients with sickle cell disease. Because Hb F level in individual sickle cells is crucial to the inhibition of cell sickling, the unique Hb F distribution may be important in determining the clinical course of this disease. © 1995 Wiley‐Liss, Inc.
Organic material concentration in auditory outer hair cells measured by laser interferometry
Authors: Laffon E. , Dilhaire S. , Aurousseau C. , Dulon D. , Claeys W. 
Published: June 2005

AbstractOuter hair cells (OHC) of the mammalian cochlea are quasicylindrical cells of different length, which play a major role in hearing at threshold. Their particular shape allows the use of a noninvasive laser interferometric technique of isolated cells in vitro in order to measure the organic material concentration (OMC), hence the density of each cell body. In most (95%) of the OHCs isolated from the same guinea pig, when the cell diameter is normalized, the results show that the cell body OMC does not vary with cell length. In different animals, the respective normalized OMC mean values can vary between 70 kg/m3 and 103 kg/m3. A few OHCs with morphological particularities often possess cell body OMCs > 103 kg/m3. The results of the interferometric measurements in isolated OHCs confirm that density variations in the cell bodies are not involved in a sound frequency coding. The in vitro OMC variotions of the OHCs could be related to the isolation procedure; however, they could also correlate with actual in vivo OMC variations. © 1995 Wiley‐Liss, Inc.
Changes in intracellular calcium concentration and pH of target cells during the cytotoxic process: A quantitative study at the single cell level
Authors: Radoškević Katarina , de Grooth Bart G. , Greve Jan 
Published: June 2005

AbstractThis study reports on the changes in intracellular calcium concentration ([Ca2+]in) and intracellular pH ([pH]in) that occur in K562 target cells during interaction with human Natural Killer (NK) cells. The data were obtained using a quantitative fluorescence microscope and fluorescent ratio probes specific for [Ca2+]in (Fura‐2‐AM) and [pH]in (BCECFAM). Results demonstrate that two types of target cell response to the attack by an NK cell can be distinguished. The target cell either dies immediately, due to the complete breakdown of the membrane impermeability, or the initial membrane damage (i.e., increased membrane permeability) is repaired and the cell “escapes” immediate death. During both responses an increase of [Ca2+]in takes place in the target cells. In the cells that die immediately, however, [Ca2+]in reaches higher levels (approximately 1,400 nM) than in the cells that restore the initial damage (approximately 700 nM). Changes in target cell [pH]in are also detected during both responses. The direction of the change (acidification or alkalinization) as well as the level of the change depend on extracellular pH ([pH]ex). Also, [pH]in remains changed during the time the cells were followed (10 min). The programming time (i.e., the time from the initiation of the cytotoxic process to the time that a change in the physiological parameter was detected) of the killing process that leads to an immediate target cell death appears to be shortest at [pH]ex 7.3–7.6 (approximately 3 min). © 1995 Wiley‐Liss, Inc.
Standardization of a flow cytometric method for measurement of low‐density lipoprotein receptor activity on blood mononuclear cells
Authors: Løhne Kari , Urdal Petter , Leren Trond P. , Tonstad Serena , Ose Leiv 
Published: June 2005

AbstractFlow cytometric methods for measurement of low‐density lipoprotein (LDL) receptor activity on peripheral blood mononuclear cells (PBMC) may be used to identify patients with familial hypercho‐lesterolemia (FH). However, cellular LDL receptor activities measured in FH heterozygotes may over‐lap with those of healthy subjects. Analytical variation is probably responsible for some of this over‐lap. We have examined several technical details that may affect analytical variation. In each analysis, we included one standard and two control cell preparations. These were cells isolated from three donors and stored in aliquots at –135°C. Use of standard cells reduced between‐series analytical variation of the controls by approximately 50%. Preincubation‐conditions used to induce the maximum number of receptors, the concentration of fluorochrome 1,1′‐dioctadecyl‐3,3,3′,3′‐tetramethylindocarbocyanine‐perchlorate (DiI)‐LDL, labelling time, and conditions during storage of labelled cells before flow cytometry were also examined in order to reduce analytical variation. Having standardized the assay, we found among 20 healthy subjects a median receptor activity of 100% vs. 51% among 26 patients who fulfilled clinical criteria for FH. However, four of the patients showed distinctly normal receptor activities, which may suggest either the presence of some other biochemical defect or that in vivo dysfunctional receptors may be measured as normal in some patients with our assay. © 1995 Wiley‐Liss, Inc.
Preferred genetic evolutionary sequences in human breast cancer: A case study
Authors: Shackney Stanley E. , Smith Charles A. , Pollice Agnese A. , Janocko Laura E. , Singh Sarita G. , Groft David W. , Brown Kathryn A. , Hartsock Robert J. 
Published: June 2005

AbstractMultiparameter flow cytometry studies were performed on the cells of an aggressive human breast cancer at the time of diagnosis and at relapse. The aneuploid cells that overexpressed large amounts of both HER‐2/neu and ras survived intensive chemotherapy and were responsible tumor relapse. At relapse, these cells were shown to overexpress simultaneously at least five oncogenes: HER‐2/neu, ras, EGF receptor, p53 and c‐myc. A partial reconstruction of the genetic evolutionary sequence in this tumor indicated that HER‐2/neu overexpression was an early step in the sequence. Subsequent HER‐2/neu overexpression, EGF receptor overexpression and p53 protein overexpression were each associated with ras overexpression. The data suggest that ploidy and oncogene overexpression cannot be used as independent clinical prognostic factors. The ability to characterize tumors according to the degree of advancement in the genetic evolutionary might serve as a basis for genetic staging for adjuvant therapy. © 1995 Wiley‐Liss, Inc.
Analytical approaches relating genetic evolutionary pathways to prognostic factors
Authors: Rohloff Albert C. , Sakach Joseph M. , Shackney Stanley E. 
Published: June 2005

AbstractHuman solid tumors accumulate multiple genetic abnormalities as they progress to advanced stages. Multiparameter flow cytometry measurements of individual cells within each tumor may be useful in describing the genetic pathways taken by individual tumors during the course of their genetic evolution. In this paper, we analyzed correlated cell‐bycell measurements of cell DNA content, HER‐2/neu protein content, and ras protein content obtained by multiparameter flow cytometry studies of primary breast cancers from 92 patients. These laboratory findings were correlated with established clinical prognostic factors for each patient at the time of diagnosis, using a stepwise multiple analysis ofvariance (MANOVA). The stepwise MANOVA successively splits a group of patients into two mutually exclusive dissimilar groups, selecting the clinical prognostic factor that is most effective in doing so. Using this criterion, formation of the first three groups that were judged most dissimilar on the cytometry parameters was based on the number of positive nodes at the time of diagnosis. We show that ploidy, HER‐2/neu protein content, and ras protein content, as measured by multiple parameter flow cytometry, are correlated with nodal status and other known clinical prognostic factors. The cellby‐cell multiparameter data suggest that for some individual tumors there are multiple genetic evolutionary pathways. Multiple genetic evolutionary pathways are also suggested by the MANOVA analysis. Focusing on the identification and analysis of genetic evolutionary pathways within individual tumors and across patients appears to offer a promising approach for defining the prognosis of early cancers. © 1995 Wiley‐Liss, Inc.