bluuebunny/crossref_metadata_2025 huggingface dataset:
Simultaneous flow cytometric detection of cellular c‐myc protein, incorporated bromodeoxyuridine, and DNA
Authors: Rosette Caridad D. , DeTeresa Patricia S. , Pallavicini Maria G.
Published: August 2005

We describe a multivariate flow cytometric technique for simultaneous analysis of specific nuclear protein, bromodeoxyuridine (BrdUrd) incorporated into DNA and DNA content in single cells in suspension. The procedure involves fixation of BrdUrd‐exposed cells with paraformaldehyde, heat denaturation of cellular DNA, followed by sequential immunochemical reactions to label incorporated BrdUrd and nuclear protein, and finally staining of total DNA with propidium iodide. The cells are analyzed flow cytometrically and multivariate data acquired in list mode to facilitate analyses of heterogeneous subpopulations. We applied this technique to measure c‐myc protein, incorporated BrdUrd, and DNA content in subpopulations present in a recombinant Chinese hamster ovary (CHO) cell line carrying approximately 800 copies of murine c‐myc sequences under control of an inducible heat shock promoter.
Proposed standard for image cytometry data files
Authors: Dean Phillip , Mascio Laura , Ow David , Sudar Damir , Mullikin James
Published: August 2005

A 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

A 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

A 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

U937 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

A 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

We 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

A 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

Several 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

By 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

The 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

Recently, 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

Graphic 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

The 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

Flow 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

The 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

Flow 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

A 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

7‐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.
Measurement of transient cDNA expression in mammalian cells using flow cytometric cell analysis and sorting
Authors: Rice Glenn C. , Pennica Diane , Borree James A. , Williams Steven R.
Published: August 2005

Generalized methods for quantitative and sensitive measurement of transient cDNA expression in mammalian cells using flow cytometry (FCM) are described. The techniques are applicable to a wide variety of cDNAs encoding intracellular or cell surface protein products through the use of immunofluorescenceor nonimmunofluorescence‐based detection methods. The methods illustrated have been optimized for sensitive detection of transfectants and efficient recovery of the encoding plasmids from the sorted cells. Expression levels and heterogeneities were compared using four methods of DNA transfer in addition to description of a novel method to optimize single copy transfer probabilities by multiparameter analysis. The overall sensitivities are compared by reconstruction and molecular cloning experiments to other methods of selection, such as immunoselection by panning. Through the measurement of multiple heterologous products per cell, or the measurement of multiple epitopes or binding sites per heterologous protein, expression levels on a single cell basis can be measured and correlated with other endpoints for various purposes. The ability to detect and recover rare clones based on a number of single and multiparameter selection criteria should significantly extend the use of transient mammalian cDNA expression methods for applications involving novel FCM‐based reporter cDNA assays and for cloning certain rare surface‐bound or secreted proteins using FCM.
Nuclear polymorphism and nuclear size in precarcinomatous and carcinomatous lesions in rat colon and liver
Authors: Zusman I. , Kozlenko M. , Zimber A.
Published: August 2005

The role of nuclear polymorphism and nuclear size in an analysis of the differences between colon and liver tumors in rats as well as in an analysis of hepatic dysplasia was studied. It was shown that colon tumors which developed following treatment of animals with a direct carcinogen alone or with a carcinogen followed by secondary bile acid were characterized by low incidence of nuclear polymorphism and by an increased nuclear size in epithelial cells. Metastatic liver tumors in rats with colon tumors were characterized by a high value for the coefficient of nuclear form polymorphism and by a significant decrease in nuclear size. Hepatic dysplasia which developed as a result of prolonged treatment with secondary bile acids was characterized by high rate of nuclear form polymorphism and by a significant increase in nuclear size. The obtained results suggest that nuclear polymorphism is dependent upon the type of tissue or organ involved in the cancerous transformation and that it may have significance as a diagnostic marker of precarcinomatous and carcinomatous lesions of digestive organs only when used in combination with other analyses.
Single‐ and double‐stranded RNA measurements by flow cytometry in solid neoplasms
Authors: El‐Naggar Adel K. , Batsakis John G. , Teague Kim , Garnsey Lisa , Barlogie Bart
Published: August 2005

We investigated the ability of single‐and double‐stranded RNA measurements to discriminate between neoplastic and non‐neoplastic solid tissues. Sixty‐one solid nonhematopoietic neoplasms, 10 reactive non‐neoplastic lesions, and 26 normal tissue samples were the test materials. Single‐stranded ribonucleic acid (s‐RNA) levels and double‐stranded ribonucleic acid (ds‐RNA) excess in these specimens were defined in relationship to normal human lymphocytes. The mean s‐RNA index in normal, reactive, benign, and diploid and aneuploid malignant tissue samples was 0.90, 1.54, 1.9, 1.2, and 2.2, respectively. For ds‐RNA, the mean excess level for normal, reactive, benign, and diploid and aneuploid malignant specimens was 8.5%, 18.5%, 51.0%, 36.0%, and 41.3%, respectively. No statistical differences in s‐RNA level were found between non‐neoplastic and neoplastic tissue samples. A significant difference in ds‐RNA excess was found between non‐neoplastic and benign, and diploid and aneuploid malignant neoplastic tissue (P<0.001). The specificity of s‐RNA level and ds‐RNA excess was 94.4% and 100%, and the sensitivity was 29.5% and 67.2%, respectively. Notably, ds‐RNA determinations identified 70.0% of the diploid neoplastic samples, in contrast to 20% by s‐RNA. Our preliminary data suggest that ds‐RNA may be a useful parameter and may complement DNA ploidy in identification of solid neoplasms, especially if the yield of intact cells is improved.
Comparison of gaussian and t‐distribution densities for modeling fluorescence dispersion in flow cytometric DNA histograms
Authors: Lampariello Francesco , Sebastiani Giovanni , Cordelli Eugenia , Spanò Marcello
Published: August 2005

In this paper, we have analyzed, on an experimental basis, the behaviour of the standardized t‐distribution with three degrees of freedom versus the gaussian model, when employed for describing the fluorescence dispersion effect in flow cytometric DNA measurements. The comparison has been performed by employing two different objective criteria based on a computer analysis for fitting DNA histograms and on a parameter related to the peak distribution shape. The results indicate that, when the peak variance is not particularly small, the two models are substantially equivalent. In the case of experimental data with very sharp peaks, the t‐distribution performs better than the gaussian density.
Coincidence in high‐speed flow cytometry: Models and measurements
Authors: Keij Jan F. , Rotterdam Ab Van , Groenewegen Ad C. , Stokdijk Willem , Visser Jan W. M.
Published: August 2005

In flow cytometry, the coincident arrival of particles becomes a major problem when high sample rates are required. For the development of our high‐speed photodamage flow cytometer (ZAPPER), it was of importance to understand the behavior of cells at flow rates of around 50,000–250,000 event/s. We developed and compared two models that describe the relation between the real cell rate and the detectable single cell rate. Both the Computer Simulation model and the Input/ Output Device model show distinct optima for the cell rate. The models were compared to measurements performed on the ZAPPER‐prototype. Fits of the two models to the experimental data were excellent for cycle times of 4 and 15 μs and acceptable for a 2 μs cycle time. A third model (Mercer WB, Rev. Sci. Instr. 37:1515–1521,1966) could be fitted to the experimental data, after the proportionality constant k was adapted to the experimental data. At a yield of detectable single cells of 70%, the maximum cell rates are 180,000, 100,000, and 40,000 cells/s for cycle times of 2, 4, and 15 μs, respectively. Based on these results we can now select an optimal cell rate for analysis and sorting based on criteria such as accepted cell loss. In addition, the advantages of reducing the cycle time can now be evaluated with respect to the costs of that modification.
Improved prognostic impact of S‐phase values from paraffin‐embedded breast and prostate carcinomas after correcting for nuclear slicing
Authors: Kallioniemi Olli‐ P. , Visakorpi Tapio , Holli Kaija , Heikkinen Asko , Isola Jorma , Koivula Timo
Published: August 2005

Nuclear debris may significantly interfere with the analysis of S‐phase fraction (SPF) from paraffin‐embedded tumors. We used a background subtraction algorithm to compensate for the effects of slicing of tumor cell nuclei during preparation of paraffin‐embedded specimens. DNA histograms were analyzed from 88 node‐negative breast and from 78 prostatic carcinomas. Median SPFs corrected for nuclear slicing were lower than un‐corrected ones in both breast cancer (7.6% vs. 5.7%) and prostate cancer (6.7% vs. 4.2%). The median SPF value in each group was used as a cut‐off point in survival studies. As compared with the uncorrected SPFs, corrected SPF levels resulted in a more significant survival difference between breast cancer patients with above and below median SPF (p = 0.0014 vs. p = 0.014) and in a higher relative risk (RR) of death (4.5 vs. 3.1). The same was true for prostate cancer survival (p < 0.0001 vs. p = 0.002) and RR (5.3 vs. 3.1). Compared with the exponential background subtraction method, the sliced nuclei correction was more reproducible and could be applied in all evaluable histograms without the risk of overcompensation. In conclusion, our results support the use of background correction with the sliced nuclei model in DNA flow cytometric studies of archival tissues.
Ki‐67 labeling in postmitotic cells defines different Ki‐67 pathways within the 2c compartment
Authors: Manoir Stanislas Du , Guillaud Philippe , Camus Emmanuel , Seigneurin Daniel , Brugal Gérard
Published: August 2005

Simultaneous quantification of DNA and Ki‐67 proliferation‐associated antigen was performed using fluorescence image cytometry. In the MCF‐7 cell line, the Ki‐67 antigen content increases during the cell cycle, and its intranuclear distribution pattern varies. Quantitative evolution of Ki‐67 content as a function of nuclear area makes it possible to define several pathways followed by cells going through the 2c compartment. 1) In some cells, the amount of Ki‐67 antigen remains constant during G1 (Ki‐67 stable pathway), and a characteristic speckled pattern can be observed. 2) In the larger fraction of cells analyzed, there is a postmitotic decrease in the Ki‐67 (Ki‐67 decrease pathway) content. In this pathway, labeling is located in the nucleoplasm in small nuclei, is located in nucleoli in intermediate‐sized nuclei, and is absent from larger nuclei (G0). A progressive increase in Ki‐67 content (Ki‐67 increase pathway) was observed from intermediate‐sized nuclei to S phase nuclei. From these results, we hypothesize that the Ki‐67 stable pathway is the G1 phase of newly formed cells going directly to S phase in local optimal conditions of growth and that Ki‐67 decrease pathway and Ki‐67 increase pathway correspond to cells whose progression to S phase is regulated by extracellular factors.
Clinical applications of cytometry: 5th annual meeting
Authors: Giorgi J. V. , Cram L. S. , Parker J. W. , Dressler L. , Kidd P. , La Via M. F.
Published: August 2005

The 5th Annual Clinical Applications of Cytometry meeting was held September 12–15, 1990 in Charlston, SC. The theme which emerged repeatedly throughout the meeting was the need to take full advantage of the quantitative power of cytometry to provide the most useful clinically relevant diagnostic and prognostic information. Greater quantitative power is based on careful and reproducible standards and quality control. The same principles, albeit kwith somewhat different approaches, apply to cell surface immunofluorescence analysis, DNA measurements, and image cytometry assessments. Monoclonal antibody probes against aoncogenes, others against lymphokines within the Golgi, and a novel fluorogenic substrate designed to quantitate the activity of a mitochondrial enzyme were exciting developments described at the meeting.
Single beam optical trapping integrated in a confocal microscope for biological applications
Authors: Visscher K. , Brakenhoff G. J.
Published: August 2005

Confocal microscopy is very useful in biology because of its three dimensional imaging capacities and has proven to be an excellent tool to study the 3D organization of, for instance, cell structures. This property of confocal microscopy makes it also very suitable for observation during guidance of the three dimensional manipulation of single cells or cell elements. Therefore we decided to integrate a confocal microscope and a single beam optical manipulator into a single instrument. The advantage of optical manipulation over mechanical techniques is that it is non‐invasive and therefore may be applied on living (micro‐) organisms and cells. The creation of an effective single beam optical trap requires the use of a high numerical aperture (N.A.) objective to focus the laser beam. In this paper we briefly discuss the vertical or axial force exerted on a sphere in a single beam trap. The axial force on a sphere placed on the optical axis, caused by reflection and refraction, is calculated applying a electromagnetic vector diffraction theory to determine the field distribution in the focal region. One of the results is that the particle also experiences a vertical trapping force towards the focusing lens when it is in the strongly convergent part of the field in addition to the known negative signed trapping force in the divergent part of the field. Further we describe an instrumental approach to realize optical trapping in which the optical trap position is controlled by moving the focusing objective only. At present we have demonstrated the practical use of the presented optical trap control on the spatial x, y, z manipulation of micro‐sized polystyrene spheres (1–7 μm) and of yeast cells.
Compliance of bacterial polyhooks measured with optical tweezers
Authors: Block Steven M. , Blair David F. , Berg Howard C.
Published: August 2005

In earlier work, a single‐beam gradient force optical trap (“optical tweezers”) was used to measure the torsional compliance of flagella in wild‐type cells of Escherichia coli that had been tethered to glass by a single flagellum. This compliance was nonlinear, exhibiting a torsionally soft phase up to 180°, followed by a torsionally rigid phase for larger angles. Values for the torsional spring constant in the soft phase were substantially less than estimates based on the rigidity determined for isolated flagellar filaments. It was suggested that the soft phase might correspond to wind‐up of the flagellar hook, and the rigid phase to wind‐up of the stiffer filament. Here, we have measured the torsional compliance of flagella on cells of an E. coli strain that produces abnormally long hooks but no filaments. The small‐angle compliance of these cells, as determined from the elastic rebound of the cell body after wind‐up and release, was found to be the same as for wild‐type cells. This confirms that the small‐angle compliance of wild‐type cells is dominated by the response of the hook. Hook flexibility is likely to play a useful role in stabilizing the flagellar bundle.
An improved hedley method for preparation of paraffin‐embedded tissues for flow cytometric analysis of ploidy and S‐phase
Authors: Heiden Thomas , Wang Naining , Tribukait Bernhard
Published: August 2005

A modification of the Hedley‐method for flow cytometric DNA analysis of paraffin‐embedded tissues is presented. Dewaxed and dehydrated tissue from paraffin blocks was incubated with subtilisin Carlsberg (pronase, Sigma protease XXIV) and then stained directly without washing and centrifugation. The loss of material was minimized, which was advantageous, for example, for the analysis of core‐biopsies, and all measured samples showed extremely low frequencies of clumped cell nuclei. This made it easier to detect polyploid nuclei and even rare nuclei of high ploidy could be identified. S‐phase analyses were more precise, since the background originating from clumped debris particles was very low. The improved method was applied to the estimation of frequencies of high‐polyploid nuclei found in various diploid, tetraploid, and aneuploid human myosarcomas of the uterus.
Flow cytometric assay for phagocytosis of human monocytes mediated via Fcγ‐receptors and complement receptor CR1 (CD35)
Authors: Andoh Akira , Fujiyama Yoshihide , Kitoh Katuyuki , Hodohara Keiko , Bamba Tadao , Hosoda Shiro
Published: August 2005

We developed a simple flow cytometric assay for phagocytosis by human monocytes that is mediated via Fcγ receptors and the complement receptor CR1 (CD35), using fluorescent latex beads carrying IgG and complement components C4b and C3b. To prepare fluorescent latex beads carrying IgG(BA), BSA‐coated latex beads (B) were incubated with diluted rabbit anti‐BSA IgG. To bind complement components, BA‐particles were incubated with whole human serum pretreated with K‐76 monocarboxylic acid (K‐76COOH). K‐76COOH inhibits the activities of C5 and factor I (12,13), resulting in the deposition of C1\documentclass{article}\pagestyle{empty}\begin{document}$ \overline{4{\rm b},2{\rm a,3b}} $\end{document} on BA‐particles (BAC1\documentclass{article}\pagestyle{empty}\begin{document}$ \overline{4{\rm b},2{\rm a,3b}} $\end{document}). Further incubation of BAC1\documentclass{article}\pagestyle{empty}\begin{document}$ \overline{4{\rm b},2{\rm a,3b}} $\end{document} with EDTA‐GVB at 37°C gave particles carrying IgG and C4b,C3b (BAC4b,3b). The C3 fragment, C3b, was confirmed to present on BAC1\documentclass{article}\pagestyle{empty}\begin{document}$ \overline{4{\rm a},2{\rm a,3b}} $\end{document} particles by SDS‐PAGE and immunoblot, and these particles were calculated to have approximately 25,000–30,000 C3b molecules per particle. To evaluate the particle attachment, the phagocytic assay was performed with 3 μM cytochalasin D treated cells. The percent cells with ingested particles and the number of ingested particles/100 cells for 60 min were estimated, being 5.1% and 5.4 for B, 12.3% and 26.7 for BA, 42.5% and 108.7 for BAC4b,3b, and 42.6% and 112.5 for BAC1\documentclass{article}\pagestyle{empty}\begin{document}$ \overline{4{\rm b},2{\rm a,3b}} $\end{document}, respectively.
Attachment of A172 human glioblastoma cells affects calcium signalling: A comparison of image cytometry, flow cytometry, and spectrofluorometry
Authors: Szöllösi Jànos , Feuerstein Burt G. , Hyun William C. , Das Manoj K. , Marton Laurence J.
Published: August 2005

The intracellular free calcium concentration ([Ca2+]i) of indo‐1 loaded A172 human glioblastoma cells stimulated by platelet‐derived growth factor (PDGF) was studied in cell suspensions by flow cytometry and spectrofluorometry and in confluent monolayers by laser image cytometry and spectrofluorometry. With all three techniques, the percentage of responsive cells, peak [Ca2+]i, and the duration of response were directly related, and the delay time was inversely related to PDGF dose. The maximum response occurred at a PDGF concentration of about 20 ng/ml. Basal and peak [Ca2+]i did not differ significantly from method to method even though different calibration procedures were used. Cells in suspension monitored by both spectrofluorometry and flow cytometry displayed significantly shorter calcium responses than attached cells. This did not appear to be a direct effect of trypsinization. Spectral analysis of indo‐1 in cytoplasm, 40% glycerol, and aqueous solutions showed significant differences in the isosbestic point and quantum efficiency. Calibration of [Ca2+]i with spectrofluorometry is more accurate using the ratio of fluorescence intensities than the fluorescence intensities measured at either 405 or 485 nm.
Simple method for quantification of fast plasma membrane movements
Authors: van Larebeke Nicolas A. F. , Bracke Marc E. , Mareel Marc M.
Published: August 2005

We present a method for the quantification of the fast plasma membrane movements that are involved in ruffling, blebbing, fast shape change, and fast translocation. The method is based on the Kontron Vidas image analysis computer program. Video images from cells viewed through an inverted microscope were transmitted to the computer. The procedure was as follows: 4 consecutive video images were averaged (image1); 28 s later a second set of 4 video images was averaged (image 2); image 2 was subtracted from image 1 and the grey level of each pixel of the resulting image was increased with 128 grey level units, resulting in the subtraction image, showing a uniform grey background speckled with brighter and darker spots corresponding to areas of movement. These spots were discriminated and turned into white objects against a black background. Interactive editing was used to delete artefacts that resulted from floating debris. The total area of the discriminated objects was measured, and the parameter motile area in μm2 per cell was calculated. We have applied our method to the study of motility induced in epithelial cell lines by the tumor promoter 12‐Otetradecanoyl‐phorbol‐13‐acetate and by epidermal growth factor.
Invasive epithelial cells show more fast plasma membrane movements than related or parental non‐invasive cells
Authors: van Larebeke Nicolas A. F. , Bracke Marc E. , Mareel Marc M.
Published: August 2005

Fast plasma membrane movements (FPMM) are involved in ruffling, blebbing, fast shape change, and fast translocation. A simple method for the quantification of FPMM was used to study the relation between FPMM and invasive capacity in five pairs of invasive and noninvasive variants from four different epithelial cell types. The human mammary cell line MCF‐7/6, the ras‐transformed dog kidney cell line ras‐MDCK, the ras‐transformed mouse mammary gland cell lines NM9‐ ras‐12 and NM‐f‐ras‐TD, and spontaneously transformed late passage mouse lens explant MLE cells, all of which were invasive in vitro, showed more FPMM in our measurements and displayed more ruffling activity on timelapse video films than the related or parental MCF‐7/AZ, MDCK‐3, NM9, and NM‐f cell lines and early passage MLE cells, none of which were invasive. Interestingly, induction of invasive capacity in MCF‐7/AZ cells by retinoic acid was accompanied by an increase in FPMM, but speed of translocation was not increased. Together these observations support the hypothesis that a certain level of FPMM is a prerequisite for invasive capacity.
Utility of light scatter in the morphological analysis of sperm
Authors: Zucker Robert M. , Perreault Sally D. , Elstein Kenneth H.
Published: August 2005

We were able to differentiate the morphologically diverse sperm nuclei of four animal species by using an Ortho flow cytometer to detect the forward light scatter from a red (helium‐neon) laser. Cytograms depicting the axial light loss and forward red scatter signals revealed unique, but reproducible, sigmoid distributions that reflected not only interspecies differences in shape and size, but variations in particle refractive index and orientation within the flow cell at the time of analysis. Consequently, we were able to use regional gating of the light scatter cytogram to minimize the influence of orientation on the resolution of the fluorescence signal. We also observed that sperm enlarging as a result of chemically induced decondensation exhibit over time a biphasic shift (increase, then decrease) in light scatter at a species‐dependent rate. These results suggest that, without any special adaptations to the flow cytometer, light‐scatter parameters can be used to discriminate morphologically different sperm, to enhance the resolution of fluorescence measurements that may otherwise be confounded by variability in radial orientation, and to detect alterations in the rate of a biochemical/biophysical process such as decondensation.