bluuebunny/crossref_metadata_2025_split huggingface dataset:
Proposed standard for image cytometry data files
Authors: Dean Phillip , Mascio Laura , Ow David , Sudar Damir , Mullikin James 
Published: August 2005

AbstractA number of different types of computers running a variety of operating systems are presently used for the collection and analysis of image cytometry data. In order to facilitate the development of sharable data analysis programs, to allow for the transport of image cytometry data from one installation to another, and to provide a uniform and controlled means for including textual information in data files, this document describes a data storage format that is proposed as a standard for use in image cytometry. In this standard, data from an image measurement are stored in a minimum of two files. One file is written in ASCII to include information about the way the image data are written and optionally, information about the sample, experiment, equipment, etc. The image data are written separately into a binary file. This standard is proposed with the intention that it will be used internationally for the storage and handling of biomedical image cytometry data. The method of data storage described in this paper is similar to those methods published in American Association of Physicists in Medicine (AAPM) Report Number 10 and in ACR‐NEMA Standards Publication Number 300‐1985.
Spatial topography of a pericentromeric region (1q12) in hemopoietic cells studied by in situ hybridization and confocal microscopy
Authors: van Dekken H. , van Rotterdam A. , Jonker R. R. , van der Voort H. T. M. , Brakenhoff G. J. , Bauman J. G. J. 
Published: August 2005

AbstractA fluorescent in situ hybridization procedure with a chromosome 1‐specific (1q12) repetitive satellite DNA probe was used to label the 1q12 regions of the chromosomes 1 in spherical and polymorphic hemopoietic cell nuclei. The entire procedure was performed in suspension to preserve nuclear morphology. The result was studied by three‐dimensional analysis, as provided by a scanning laser confocal microscope. The 1q12 regions of chromosome 1 were measured to be closely associated with the nuclear envelope in isolated nuclei of unstimulated diploid human lymphocytes. The relative positions to each other in the periphery of these spherical nuclei could not be distinguished from a random distribution pattern. In the diploid and tetraploid polymorphic nuclei of cells of the promyelocytic leukemia cell line HL60 these pericentromeric sequences were also associated with the nuclear surface.
Three‐dimensional reconstruction of pericentromeric (1q12) DNA and ribosomal RNA sequences in HL60 cells after double‐target in situ hybridization and confocal microscopy
Authors: van Dekken H. , van der Voort H. T. M. , Brakenhoff G. J. , Bauman J. G. J. 
Published: August 2005

AbstractA fluorescent in situ hybridization procedure was applied to simultaneously label intranuclear pericentromeric (1q12) sequences of the chromosomes 1 and cytoplasmic ribosomal RNA sequences in whole cells of the promyelocytic HL60 cell line. For this purpose biotinated chromosome 1‐specific (1q12) repetitive satellite DNA and 28S ribosomal ssRNA probes were used. The entire procedure was performed in suspension to preserve nuclear morphology. The result was studied by three‐dimensional analysis, as provided by a scanning laser confocal microscope. The intracellular positions of both cytoplasmic rRNA and intranuclear centromere 1 DNA could easily be distinguished. This approach could be useful as a framework for the study of the 3‐D localization of genes and gene transcripts.
Synchronisation of cancer cell lines of human origin using methotrexate
Authors: Sen Soumitra , Erba Eugenio , D'Incalci Maurizio 
Published: August 2005

AbstractU937 human histiocytic lymphoma cell line and SW626 ovarian carcinoma line of human origin were synchronised using very low, nontoxic concentrations (0.04–0.08 μM for 16–24 h) of methotrexate (MTX) under standard culture conditions. Satisfactory synchrony was achieved to study S phase events. Various kinetic behaviours and biological properties of the synchronised cells are considered for characterisation of the system. MTX‐synchronisation was compared with that induced by aphidicolin (APC) alone and by serum deprivation and APC. In some cancer cell lines MTX appears to be the best choice for obtaining highly synchronised cell populations without cytotoxicity or physiological perturbations.
Flow cytometric assay for the measurement of human bone marrow phenotype, function and cell cycle
Authors: Lund‐Johansen Fridtjof , Bjerknes Robert , Laerum Ole Didrik 
Published: August 2005

AbstractA flow cytometric assay for the measurement of human bone marrow and blood leukocyte antigen expression, phagocytosis, and proliferation is described. Subpopulations of leukocytes were identified by their light scatter characteristics, and the expression of a myeloid differentiation antigen (designated CDw65) determined following incubation with CDw65 specific fluorescein‐isothiocyanate (FITC) conjugated monoclonal antibodies (VIM2). Incubation of leukocytes with ethidium monoazide (EMA) labeled Candida albicans followed by staining with FITC conjugated VIM2 allowed the combined determination of cellular CDw65 expression and phagocytic capacity. In addition, immunostained leukocytes were fixed, and their DNA labeled with propidium iodide (PI), before CDw65 expression was measured for cells in different phases of the cell cycle. The method allows evaluation of phenotypic and functional heterogeneity, as well as cell cycle parameters, within subpopulations of cells during hematopoietic differentiation.
Flow cytometric analysis of DNA content and keratins by using CK7, CK8, CK18, CK19, and KL1 monoclonal antibodies in benign and malignant human breast tumors
Authors: Ferrero Magali , Spyratos Frédérique , Le Doussalg Viviane , Desplaces Andrée , Rouesse Jacques 
Published: August 2005

AbstractWe have used a double‐labelling flow cytometry analysis of keratin (CK) and DNA in breast cancer. Five monoclonal anti‐keratin antibodies were tested: KL1 recognizing Mr 55,000–57,000 keratins, and “anti‐glandular epithelia,” LE41, RGE‐53, and LP2K specific for CK n° 7, 8, 18, and 19 of Moll's classification, respectively. Flow cytometric (DNA‐CK) analysis was performed on 10 benign and 19 malignant human breast tumors. All the benign tumors were diploid and 63% of the malignant tumors were aneuploid. This technique permits the analysis of DNA in the epithelial fraction alone. In aneuploid tumors, gating the DNA‐keratin‐positive population allowed accurate DNA analysis without interference due to debris background and non‐epithelial cells. Moreover, double‐labelling using the CK19 antibody gave a better identification of near‐diploid tumors. An enhancement of keratin expression in malignant tumors was observed with CK 19 (P < 0.001), KL1 (P < 0.01), CK 8 (P < 0.05), and CK18 (n.s.) compared to benign tumors. The comparison of keratin expression in aneuploid and diploid malignant tumors revealed reduced CK8, CK18, and CK19 in the former.
Inorganic phosphors as new luminescent labels for immunocytochemistry and time‐resolved microscopy
Authors: Beverloo H. B. , van Schadewijk A. , van Gelderen‐Boele S. , Tanke H. J. 
Published: August 2005

AbstractA new strongly luminescent marker consisting of inorganic crystals is described for time‐resolved microscopy. These crystals, known as phosphors, show delayed luminescence, unlike prompt fluorescent labels such as FITC, TRITC and phycobiliproteins, and are therefore potentially suitable for time‐resolved microscopy. The luminescence of these phosphors is strong and non‐fading in comparison to FITC/TRITC, and not significantly influenced by pH or temperature. The phosphor yttriumoxisulfide activated with europium emits maximally at 620 nm with a typical half lifetime of approximately 700 μsec, upon excitation with near ultraviolet light (360 nm).Phosphors for immunocytochemical staining were made by ball milling and were stabilized in suspension with polycarboxylic acids. Proteins such as avidin, protein A or immunoglobulins were allowed to adsorb to the surface of the phosphors. The immunocytochemical properties of the conjugates were evaluated in a model system of latex beads with defined surface antigens and in a cellular system containing fixed human lymphocytes or erythrocytes. Specific cytochemical staining was observed in suspension as well as on glass slides. A specially constructed time‐resolved microscope was used to suppress the fast decaying fluorescence, thereby permitting visualization of the specific, slowly decaying luminescence of the phosphor label without the necessity of integration. Finally, the use of multiple phosphors with different kinetic and spectral characteristics for multiparameter studies is indicated.
Flow cytometric analysis of G1‐ and G2/M‐phase subpopulations in mammalian cell nuclei using side scatter and DNA content measurements
Authors: Nüsse Michael , Beisker Wolfgang , Hoffmann Christine , Tarnok Attila 
Published: August 2005

AbstractSeveral subcompartments of the cell cycle in addition to the G1‐, S‐, and G2‐phases usually observed were identified by simultaneous flow cytometric measurements of ethidium bromide fluorescence and side scatter intensity of cell nuclei. Metaphase cells and very early G1‐phase cells (G1A) with low side scatter intensities were discriminated from interphase cells with high side scatter intensities. The reason for the various side scatter intensities was found to be the different structure of metaphase cells and early G1‐phase cells due to chromatin condensation as shown by sorting of the respective cell nuclei. The G1A‐phase could further be subdivided into two compartments with very low side scatter (G1A1) and intermediate side scatter (G1A2) intensities. Using partially synchronized cells the duration of these subcompartments of the G1‐phase could be estimated. The durations of G1A1‐ and G1A2‐phases were found to be about 10 min and 20 min, respectively, compared to the total duration of the G1‐phase of about 3 h. Additional flow cytometric measurements of side scatter intensities of cell nuclei provide therefore further information on subcompartments of the G1‐ and G2/M‐phases.
Increasing sensitivity of the Ortho analytical cytofluorograph by modifying the fluid system
Authors: Zucker Robert M. , Elstein Kenneth H. , Gershey Edward L. , Massaro Edward J. 
Published: August 2005

AbstractBy adding Teflon tubing to the effluent line of the Ortho 50H analytical cytofluorograph, we were able to increase the sensitivity of fluorescence and scatter detection threefold without compromising resolution. By increasing sheath backpressure (resistance), the additional tubing increases particle residence time within the detection area and thereby increases the total photon emission density per particle. In addition, the longer, wider tubing dampens the stream‐destabilizing effects of minor obstructions in or movement of the effluent line. This increase in sensitivity and stability is desirable in applications requiring detection of particles exhibiting low‐level fluorescence.
Quantification of renal Na‐K‐ATPase activity by image analysing system
Authors: Laborde K. , Bussieres L. , De Smet A. , Dechaux M. , Sachs C. 
Published: August 2005

AbstractThe localisation of renal Na‐K‐ATPase activity along the rat nephron by a cytochemical method, and its quantification by an image analysis system, are described in this paper. Frozen kidney sections were exposed to a trapping agent, the lead ammoniac‐citrate‐acetate complex (LACA), and to all the substrates necessary to the enzyme acitvity. The absorbance of the histochemical reaction product (precipitated in situ), proportional to the enzymatic activity, was then measured through the analysis of the grey levels of the transmitted image of the kidney section. This method was both sufficiently sensitive and technically simple to permit measurements of the enzyme in large numbers of tubules and to determine its activity in each region of the nephron. The Na‐K‐ATPase activity has been determined in the proximal convoluted tubule (PCT), the medullary thick ascending limb of the Henle's loop (mTAL), and the distal convoluted tubules (DCT) of the rat nephron. The Na‐K‐ATPase distribution shows an activity per millimeter tubule length higher in the DCT than in the mTAL and the PCT: 1,406 ± 33, 823 ± 64, and 350 ± 71 pmoles Pi/tubule mm/h, respectively. In conclusion, the described method allows the segmental quantification of Na‐K‐ATPase activity at a cellular level and offers a precise approach to the analysis of this enzyme along the length of nephrons.
A method for calibration of flow cytometric wavelength shift fluorescence measurements
Authors: Kachel V. , Kempski O. , Peters J. , Schödel F. 
Published: August 2005

AbstractRecently, new fluorescent dyes have been introduced into flow cytometry which alter their spectral characteristics when changes occur in certain cell features, e.g., intracellular pH or calcium ion concentration. Such changes may be determined by measuring the fluorescence intensity ratio in two different wavelength ranges (5). Here a new method is described, which simplifies the use of steadily flowing fluids for calibration. The pulse electronics of a flow cytometer cannot process the static fluorescence signals of a streaming fluid. If however, the exciting or emitted fluorescence light of a calibration fluid is made pulsating, the flow cytometer electronics can evaluate those pulses. The new calibration procedure uses measurement of two wavelength windows shown in a two‐parameter display to generate an absolute calibration scale. Measurement of the spectral shift in calibration fluids under identical instrumental settings provides absolute values that measurements of intracellular concentrations can be referred to.
Macintosh graphics for the EPICS flow cytometer user
Authors: Cameron Mark J. 
Published: August 2005

AbstractGraphic options for the EPICS flow cytometer user have been restricted in the past to software written specifically for the analysis and graphics of flow cytometric data. The software is limited to only a few graphic presentation styles. The technique described will allow the EPICS user to translate histogram files into text files that can be used in an alternative computer format.
A new class of reversible cell cycle inhibitors
Authors: Hoffman Benjamin D. , Hanauske‐Abel Hartmut M. , Flint Alan , Lalande Marc 
Published: August 2005

AbstractThe effects of three compounds on the cell cycle of HL‐60 promyeloid leukemia cells has been examined. Ciclopirox olamine, an antifungal agent, and the compound Hoechst 768159 reversibly block the cell cycle at a point occurring roughly 1 h before the arrest mediated by aphidicolin, an inhibitor of DNA polymerase á activity, which acts in early S phase. Similar results are also obtained with the compound mimosine, a plant amino acid. Based on these data, it is concluded that all three agents inhibit cell cycle traverse at or very near the G1/S phase boundary and identify a previously undefined reversible cell cycle arrest point.
Flow cytometric measurement of cell cycle kinetics in rat Walker‐256 carcinoma following in vivo and in vitro pulse labelling with bromodeoxyuridine
Authors: Fogt Franz , Wan Jennifer , O'Hara Carl , Bistrian Bruce R. , Blackburn George L. , Istfan Nawfal W. 
Published: August 2005

AbstractFlow cytometric measurements of total DNA content, cell cycle distribution, and bromodeoxyuridine (BrdUrd) uptake were made in rat Walker 256 carcinoma cells. After both in vivo and in vitro pulse labelling with BrdUrd, Walker‐256 tumor cells were stained with propidium iodide (PI) to estimate the total DNA content and a monoclonal antibody against BrdUrd to estimate the relative amount of cells in S phase. BrdUrd‐labelled single cell suspensions were harvested at different time intervals to determine the movement of these cells within the cell cycle. To increase BrdUrd uptake, fluorodeoxyuridine (FDU), a thymidine antagonist, was also applied in vivo and in vitro. The results indicated exponential growth characteristics for this tumor between days 5and 8 after implantation. Tumor doubling times, derived from changes in tumor volume in vivo and from the increase in cell number in vitro were similar. The mean time for DNA synthesis was estimated from the relative movement of BrdUrd‐labelled cells towards G2. The percent of cells labelled with BrdUrd and the DNA synthesis time were similar regardless of the mode of BrdUrd administration. This study demonstrates that BrdUrd labelling of rat Walker‐256 carcinoma cells in vitro yields kinetic estimates of tumor proliferation during exponential growth similar to those with the administration of BrdUrd in the intact tumor‐bearing rat.
An innovation in flow cytometry data collection and analysis producing a correlated multiple sample analysis in a single file
Authors: Robinson J. Paul , Durack Gary , Kelley Steve 
Published: August 2005

AbstractThe problems associated with rapid analysis and interpretation of data from multicolor immunofluorescence panels have been a formidable barrier to their routine use. Using present flow cytometry concepts, a panel of 11 tubes each containing multiple phenotypic markers or controls requires postdata acquisition manipulation of many multiparameter histogram and listmode files. We have developed a method that compresses all of the information from such a panel into a single listmode data file during run time. A single data file is used to record the entire phenotypic analysis for a particular patient or series within an experiment. This is accomplished by the incorporation of a tube identifier parameter (TIP) as well as the fluorescence and light scatter parameters normally collected. The TIP can then be used for gating discrimination of any tube or set of tubes within a panel. When the TIP is correlated with the PRISM parameter the entire patient phenotypic image can be represented within a single two‐parameter histogram we have called a phenogram. This phenogram can be generated in real time, providing on‐line preprocessing of a complex multicolor experiment. By examining the image created by the phenogram it is possible to rapidly flag abnormalities such as incorrect gating. This procedure was carried out on an EPICS Elite flow cytometer in its standard configuration with the addition of hardware to provide an input for the TIP.
Flow cytometric analysis of DNA stemline heterogeneity in primary and metastatic breast cancer
Authors: Beerman Henk , Smit Vincent T. H. B. M. , Kluin Philip M. , Bonsing Bert A. , Hermans Jo , Cornelisse Cees J. 
Published: August 2005

AbstractFlow cytometric DNA‐ploidy analysis was used to investigate intratumor DNA stemline heterogeneity in primary breast carcinomas and lymph node metastases (LNM). The study was done in tumor specimens from 44 patients 35 of whom had LNM. In all, measurements were done in 214 different samples of primary tumors and 211 lymph nodes. Sixty‐one percent (27/44) of the primary tumors were found to have multiple DNA aneuploid stemlines when the data of the separate samples per tumor (mean 4.9) were compared. Only five of 44 (11%) primary tumors were DNA diploid; two of these had DNA aneuploid metastases. Statistical analysis of these results indicated that, on average, four samples are needed for reliable determination of the DNA ploidy status of primary tumors by flow cytometry. In the majority of the cases (26/35), distinct tumor DNA stemlines found in LNM were also present in the primary tumor, which suggests that the generation of DNA ploidy diversity may have taken place prior to metastasis. Multiploidy was not related to tumor size but, particularly for LNM, was significantly correlated with age (r = 0.40, P = 0.02). The results of this study support the view that breast cancer is an extremely heterogeneous disease and that underestimation of this factor might account for the disagreement in literature about the prognostic value of DNA ploidy determinations.
Progesterone receptor detection and quantification in breast tumors by bivariate immunofluorescence/DNA flow cytometry
Authors: Remvikos Yorghos , VuHai Maithu , Lainé‐Bidron Christine , Jollivet André , Magdelénat Henri 
Published: August 2005

AbstractA method was developed for the detection of progesterone receptors (PgR) by flow cytometry (FCM) in cell suspensions obtained from mechanically dispersed fragments of operated breast cancers. Two monoclonal antibodies were tested for sensitivity and specificity on four breast cancer cell lines of known PgR expression and a calibration curve thus established. A simple procedure was used to calculate the level of PgR expression, taking into account the relative displacement of total cellular fluorescence compared to nonspecific fluorescence for each sample and the average DNA content of the cells derived from the corresponding histograms. The PgR‐speciflc immunofluorescence of the tumor specimens measured in arbitrary units (channels) was then transformed to fmoles/mg DNA by comparison with the calibration curve. The FCM‐derived results were compared with those of a conventional immunoenzymatic PgR assay on 30 surgical samples. PgR content ranged from 10 to 22,000 fmoles/mg DNA and linear regression analysis yielded a good correlation (r = 0.86). With a threshold of positivity of 300 fmoles/mg DNA, the two methods concurred for 28 of 30 tumors (93%). Nine specimens were analyzed repeatedly, showing good reproducibility. This method could prove to be more useful than the biochemical assays on homogenates, since it allows the simultaneous analysis of receptor expression in individual cells and of DNA index (ploidy).
Simultaneous assessment of chromatin structure, DNA content, and antigen expression by dual wavelength excitation flow cytometry
Authors: Stokke Trond , Holte Harald , Erikstein Bjørn , Davies Catharina De L. , Funderud Steinar , Steen Harald B. 
Published: August 2005

Abstract7‐aminoactinomycin D (7‐AMD) efficiently discriminates between cells in the G0 and G1 phases of the cell cycle (Stokke et al., Cancer Res. 48:6708, 1988). The fluorescence and light scatter of cells stained with 7‐AMD, Hoechst 33258 (H33258), and fluorescein isothiocyanate (FITC)‐labeled antibodies were measured by dual wavelength excitation flow cytometry (488 nm, ultraviolet). The H33258 fluorescence was found to reflect DNA content in the presence of 7‐AMD, although energy transfer caused an approximately 50% reduction in H33258 fluorescence intensity. However, energy transfer was more pronounced in dead cells, permitting exclusion of such cells during analysis. The G0, G1, S, and G2 phases of the cell cycle could be identified in the 7‐AMD versus H33258 fluorescence histograms, as was demonstrated with mitogen‐stimulated B lymphocytes and a mixture of unstimulated B lymphocytes and a proliferating B‐cell line. One hour fixation with paraformaldehyde was compatible with prefixation labeling of surface antigens with indirectly FITC‐labeled antibodies as well as postfixation labeling of intracellular antigens. Studies of expression of some surface and nuclear activation‐associated antigens confirmed that cell cycle‐resolved antigen expression and the time course of appearance of such antigens could be assessed accurately. Phycoerythrin could be used to label a second antigen.