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PubMed2001

Morphometry of nucleoli and expression of nucleolin analyzed by laser scanning cytometry in mitogenically stimulated lymphocytes.

BACKGROUND: Various attributes of nucleoli, including abundance of the nucleolar product (rRNA), correlate with cell-proliferative status and are useful markers for tumor diagnosis and prognosis. However, there is a paucity of methods that can quantitatively probe nucleolus. The aim of the present study was to utilize the morphometric capacity of the laser scanning cytometer (LSC) to analyze nucleoli and measure expression of the nucleolar protein nucleolin (NCL) in individual cells and correlate it with their state of proliferation. MATERIALS AND METHODS: Human lymphocytes were mitogenically stimulated, and at different time points their nucleoli were detected immunocytochemically using NCL Ab. The frequency of nucleoli per nucleus, their area, and the level of expression of NCL, separately in the nuclear and nucleolar compartments, were estimated in relation to the G(0) to G(1) transition and the cell cycle progression. RESULTS: During the first 24 h of stimulation, when the cells underwent G(0) to G(1) transition, their RNA content was increased nearly 8-fold, the level of NCL per nucleus also increased 8-fold, the NCL per nucleolus increased 12-fold, nucleolear area increased 3-fold, and NCL/nucleolar area increased nearly 4-fold. During the subsequent 24-48 h of stimulation, when cells were progressing through S, G(2), and M and reentering the next cycle, the number of nucleoli per nucleus was increased and a massive translocation of NCL from nucleoli to nucleoplasm was observed; its overall level per nucleus, however, still remained high, at 6-fold above of that of G(0) cells. CONCLUSIONS: While high expression of NCL in the nucleolar compartment correlates with the rate of rRNA accumulation in the cell and is a sensitive marker of the G(0) to G(1) transition, the cells progressing through the remainder of the cycle are better distinguished from G(0) cells by high overall level of NCL within the nucleus. Such an analysis, when applied to tumors, may be helpful in obtaining the quantitative parameters related to the kinetic status of the tumor-cell population and tumor prognosis. The capability of LSC to measure the protein translocation between nucleolus and nucleoplasm can be used to study the function and regulatory mechanisms of other proteins that reside in these compartments.

Cell Nucleolus↗
PubMed2001

Bivariate analysis of cellular DNA versus RNA content by laser scanning cytometry using the product of signal subtraction (differential fluorescence) as a separate parameter.

BACKGROUND: The cytometric methods of bivariate analysis of cellular RNA versus DNA content have limitations. The method based on the use of metachromatic fluorochrome acridine orange (AO) requires rigorous conditions of the equilibrium staining whereas pyronin Y and Hoechst 33342 necessitate the use of an instrument that provides two-laser excitation, including the ultraviolet (UV) light wavelength. METHODS: Phytohemagglutinin (PHA)-stimulated human lymphocytes were deposited on microscope slides and fixed. DNA and double-stranded (ds) RNA were stained with propidium iodide (PI) and protein was stained with BODIPY 630/650-X or fluorescein isothiocyanate (FITC). Cellular fluorescence was measured with a laser scanning cytometer (LSC). The cells were treated with RNase A and their fluorescence was measured again. The file-merge feature of the LSC was used to record the cell PI fluorescence measurements prior to and after the RNase treatment in list mode, as a single file. The integrated PI fluorescence intensity of each cell after RNase treatment was subtracted from the fluorescence intensity of the same cell measured prior to RNase treatment. This RNase-specific differential value of fluorescence (differential fluorescence [DF]) was plotted against the cell fluorescence measured after RNase treatment or against the protein-associated BODIPY 630/650-X or FITC fluorescence. RESULTS: The scattergrams were characteristic of the RNA versus DNA bivariate distributions where DF represented cellular ds RNA content and fluorescence intensity of the RNase-treated cells, their DNA content. The distributions were used to correlate cellular ds RNA content with the cell cycle position or with protein content. CONCLUSIONS: One advantage of this novel approach based on the recording and plotting of DF is that only the RNase -specific fraction of cell fluorescence is measured with no contribution of nonspecific components (e.g., due to the emission spectrum overlap or stainability of other than RNA cell constituents). Another advantage is the method's simplicity, which ensues from the use of a single dye, the same illumination, and the same emission wavelength detection sensor for measurement of both DNA and ds RNA. The method can be extended for multiparameter analysis of cell populations stained with other fluorochromes of the same-wavelength emission but targeted (e.g., immunocytochemically) for different cell constituents.

Cell Cycle↗
PubMed2000

Translocation of Bax to mitochondria during apoptosis measured by laser scanning cytometry.

BACKGROUND: During induction of apoptosis, the pro-apoptotic member of the Bcl-2 protein family (Bax) undergoes translocation to the mitochondria. The translocation, which leads to accumulation of Bax in the mitochondrial intermembrane space, appears to be the critical event determining release of cytochrome c to cytosol: the latter event triggers the irreversible steps of apoptosis, namely, the activation of caspases and the initiation of the degradation of many proteins. The aim of this study was to utilize the morphometric capabilities of the laser scanning cytometer (LSC) and adapt this instrument to detect and measure in situ the process of translocation of Bax to mitochondria. METHODS: Human breast carcinoma MCF-7 cells growing on microscope slides were treated with the DNA topoisomerase I inhibitor, camptothecin (CPT). CPT is known to induce apoptosis preferentially of S-phase cells. The cells were fixed and permeabilized on the slides, their DNA was stained with propidium iodide (PI), Bax was detected immunocytochemically with the fluoresceinated antibody, and red and green fluorescence emission was measured by the LSC. RESULTS: Prior to induction of apoptosis, Bax was uniformly and diffusely dispersed in the cell nucleus and cytoplasm. Its translocation and accumulation in mitochondria in cells undergoing apoptosis were detected and measured by the LSC as the increase in intensity of maximal pixel of Bax immunofluorescence. Bivariate analysis of DNA content versus maximal pixel of Bax fluorescence revealed that the CPT-induced Bax translocation into mitochondria was preferential to S-phase cells. Total cellular Bax immunofluorescence measured by flow cytometry, however, was increased in all phases of the cycle without a preference to S-phase cells. CONCLUSION: Changes in abundance and localization of particular proteins that undergo translocation within the cell, leading to their altered local density, may be conveniently detected by the LSC by taking advantage of its morphometric capabilities. Measurement of total cellular Bax immunofluorescence by flow cytometry combined with analysis of its translocation by LSC revealed that apoptosis of S-phase cells induced by CPT was unrelated to overall Bax abundance per cell but correlated with its accumulation in mitochondria.

Apoptosis↗
PubMed2001

Effect of chemicals on the duration of male meiosis in mice detected with laser scanning cytometry.

Aneuploidy studies in sperm such as the sperm-FISH assay require a precise knowledge of the duration of spermatogenesis, especially of the meiotic stages. This is important in order to sample sperm from the epididymis at appropriate intervals after animal treatment. However, aneugens may delay the cell cycle. The progression from meiotic divisions to epididymal sperm was determined by labelling the last S-phase before meiosis with the thymidine analogue 5-bromo-2'-deoxyuridine (BrdU) and treating the animals 13 days later with the test chemicals. In a time frame of 20--24 days after treatment, BrdU-containing sperm were identified with a FITC-labelled anti-BrdU antibody and green fluorescent sperm were scored with a laser scanning cytometer (LSC). We studied the effects of the chemicals acrylamide, colchicine, diazepam, griseofulvin, taxol, thiobendazole, trichlorfon and vinblastine on the duration of meiotic divisions in male mice. Colchicine treatment prolonged the duration of meiotic divisions by about 48 h. On days 21 and 22, the frequencies of BrdU-labelled sperm in the colchicine group were 11.7 and 9.4%, respectively, while they were 28.4 and 30.6%, respectively, in the concurrent controls (P > 0.01). On day 24 after treatment, the frequency of labelled sperm in the colchicine group reached the control level. Etoposide treatment resulted in an elevation of BrdU-labelled sperm at 23 rather than 22 days. The other chemicals showed no significant effect of prolonging meiotic cell cycle progression. On the basis of the colchicine and etoposide data, it is suggested that the effect of a chemical on the meiotic cell cycle progression is determined first in order to chose the appropriate sperm sampling time to detect aneuploidy induction.

Animals↗
PubMed2002

Cell array coupled with laser scanning cytometry allows easy analysis of changes in cyclin expression during the cell cycle. An application of cell array system.

To assess the potential of cell array technology, a cell array slide with 50 spots was designed specifically for automated analysis of changes in expression of cyclins A and B1 during the cell cycle at the cellular level. Cells harvested every 1 hour from 0 through 23 hours after synchronization by mitotic selection were spotted in duplicate on a glass slide. Each slide contained 48 spots representing 24 different cell cycle phases, and the remaining two spots were peripheral blood lymphocytes, which were included as negative control. This cell array provided temporal and spatial information related to changes in cyclin expression during the cell cycle in a single experiment. The present study indicates that expression analysis by cell array is novel approach for cell cycle studies. Furthermore, sophisticated multiplexed cell array technology has great potential for analyses of expression of specific genes during diverse cellular events at the cellular level.

Cell Cycle↗
PubMed2001

Use of flow and laser scanning cytometry to study mechanisms regulating cell cycle and controlling cell death.

Applications of FC and more recently LSC in the fields of cell cycle progression and cell death already have revealed a wealth of knowledge about mechanisms involved in regulation of these pathways. The cytometry instrumentation and the associated methodologies complement tools of molecular biology by providing information that is unavailable from the latter alone. The use of cytometry side by side with classic molecular biology methods offers the means for deeper exploration of these mechanisms at molecular and cellular levels. Recent years brought the availability of new cytometric probes that sense the functional changes in the cell, such as chemical modifications (phosphorylation, acetylation, ubiquitinylation, and so forth) of the regulatory molecules, or their translocations between different cell compartments. Such probes will be particularly useful in mechanistic studies on cell cycle and cell death. One expects that the progress in these fields will expand at a continuously accelerating pace, propelled by development of new methodologies and probes. One of the dominating motors for this progress will be further development of cytometry instrumentation and the applicability of this instrumentation, methods, and probes.

Apoptosis↗
PubMedDate not supplied

Laser technologies in toxicopathology.

One of the main concepts in toxicology and risk assessment is the identification of compounds with the least toxicity, gaining increased understanding of the underlying mechanisms of efficacy and toxicity so as to accelerate the early selection of compounds for development. For this purpose, "cutting-edge" technologies, such as flow cytometry (FC), laser scanning cytometry (LSC) and confocal laser scanning microscopy (CLSM), have proved to be valuable tools. FC, LSC and CLSM have been successfully applied in a wide range of areas within toxicology and research including genetics, reproduction, dermatology, pathology and target organ toxicity. The scope of this paper is to give a short overview of the usefulness of the different laser applications. Specific examples of the impact of these technologies will be presented or can be found in the references. Flow cytometry methods have been successfully applied in immunophenotyping, micronuclei scoring, polyploidy determination, apoptosis and cell cycle evaluation, cell proliferation and quantification. A three-parameter FC method for the analysis of testicular toxicity has also been established as an alternative to traditional histopathological methods. This method allows a large number of cells to be analysed in a short time and provides quantitative values to evaluate testicular damage in the rat. Laser scanning cytometry has been used in our unit for rat blood cell immunophenotyping, tumor proliferation, apoptosis and cell cycle analysis on minipig and rat skin and cardiac cells identification. The wide range of applications that can be applied with the LSC shows the enormous potential of this technology in research and development. Confocal laser scanning microscope was used in our laboratory, in collaboration with the research department, to investigate the mechanisms underlying hepatic lesions found in dogs, to detect fibrinogen influx into rat lung, to explore the mechanism of eye toxicity and to quantify dopaminergic fibers in brain sections. Integrating these technologies within discovery pathology allowed us to understand disease processes with respect to their development and subsequent consequences. It contributes to descriptive pathologic diagnostic and allows a productive interaction with research and development. These technologies offer a range of novel applications and have been shown to be useful tools in terms of specificity, sensitivity, reliability, rapidity and quantification. Expertise in cutting-edge technologies, pathology and cell and molecular biology is essential to a successful and flexible interaction across all therapeutic areas in drug discovery.

Animals↗
PubMed2003

CytometryML, an XML format based on DICOM and FCS for analytical cytology data.

BACKGROUND: Flow Cytometry Standard (FCS) was initially created to standardize the software researchers use to analyze, transmit, and store data produced by flow cytometers and sorters. Because of the clinical utility of flow cytometry, it is necessary to have a standard consistent with the requirements of medical regulatory agencies. METHODS: We extended the existing mapping of FCS to the Digital Imaging and Communications in Medicine (DICOM) standard to include list-mode data produced by flow cytometry, laser scanning cytometry, and microscopic image cytometry. FCS list-mode was mapped to the DICOM Waveform Information Object. We created a collection of Extensible Markup Language (XML) schemas to express the DICOM analytical cytologic text-based data types except for large binary objects. We also developed a cytometry markup language, CytometryML, in an open environment subject to continuous peer review. RESULTS: The feasibility of expressing the data contained in FCS, including list-mode in DICOM, was demonstrated; and a preliminary mapping for list-mode data in the form of XML schemas and documents was completed. DICOM permitted the creation of indices that can be used to rapidly locate in a list-mode file the cells that are members of a subset. DICOM and its coding schemes for other medical standards can be represented by XML schemas, which can be combined with other relevant XML applications, such as Mathematical Markup Language (MathML). CONCLUSIONS: The use of XML format based on DICOM for analytical cytology met most of the previously specified requirements and appears capable of meeting the others; therefore, the present FCS should be retired and replaced by an open, XML-based, standard CytometryML.

Computer Communication Networks↗
PubMedDate not supplied

Effects of tyrosine kinase signaling inhibition on survival after cecal ligation and puncture in diet-restricted mice.

BACKGROUND: Malnutrition impairs host immunity, resulting in high mortality and morbidity due to infections. Phosphorylation of protein tyrosine kinase (PTK) is a key step in the signaling of many cellular functions, including immune cell functions. Malnutrition may affect this signaling in response to surgical insults. The aim of this study was to examine the effects of PTK inhibition on mortality in ad libitum and in diet-restricted mice after cecal ligation and puncture (CLP). Moreover, tyrosine phosphorylation of peritoneal cells from these animals was evaluated. METHODS: Survival study: Mice (n = 45) received chow, 146 g/kg per day (ad libitum) or 36.5 g/kg per day (diet-restricted), for 7 days. Two hours before CLP, one-half the mice in each group were given a tyrosine kinase inhibitor, AG 556 (3.0 mg/kg i.p.), and the others received vehicle. Survival was observed up to 7 days after CLP. Effects of AG 556 on survival with a lesser degree of malnutrition (chow 73 g/kg per day) were also examined (n = 41). Measurement of tyrosine phosphorylation: mice (n = 20) were assigned to the ad libitum and diet-restricted (chow 36.5 g/kg per day) groups. Peritoneal cells were harvested either before or 2 hours after glycogen injection. Glycogen treatment elicits polymorphonuclear neutrophil influx into the peritoneal cavity. The cells were incubated with or without N-formyl-methionyl-leucyl-phenylalanine (fMLP). Tyrosine phosphorylation in the cells was examined using flow cytometry, laser scanning cytometry, and Western blotting. RESULTS: Diet restriction significantly reduced survival compared with the ad libitum group. AG 556 treatment decreased the survival of ad libitum, but not in diet-restricted mice in both survival experiments. Stimulation of peritoneal cells with fMLP increased tyrosine phosphorylation in the ad libitum group (23% increase before glycogen and 18% after glycogen), but not in the diet-restricted group (-9% before glycogen and 3% after glycogen). CONCLUSIONS: Inhibition of tyrosine kinase signaling impairs the ability of a well-nourished host to survive CLP-induced sepsis, while having no effects on survival in diet-restricted mice. Peritoneal cells from diet-restricted animals are unable to increase PTK phosphorylation in response to stimulation, which may be the mechanism underlying impaired host defense during malnutrition.

Animals↗
PubMed2006

Cytological response of hemocytes in the European flat oyster, Ostrea edulis, experimentally exposed to mercury.

Molluscs bivalves have been widely used as bioindicators to monitor contamination levels in coastal waters. In addition, many studies have attempted to analyze bivalve organs, considered pollutant-targets, to understand the bio-accumulation process and to characterize the effects of pollutants on the organisms. Here we analyzed the effects of mercury exposure on flat oyster hemocytes. Optical and electronic microscope procedures were used to characterize hemocyte morphology. In addition, cell solutions treated with acridine orange were analyzed by flow cytometry and laser scanning cytometry in order to evaluate the variations of cytoplasmic granules (red fluorescence, ARF) and cell size (green fluorescence, AGF) of hemocyte populations over time. Light and electron microscopical studies enabled us to differentiate four hemocyte subpopulations, agranulocytes (Types I and II) and granulocytes (Types I and II). Slight morphological differences were observed between control and Hg-exposed cells only in granulocytes exposed to Hg for 30 days, where condensed chromatin and partially lysed cytoplasmic regions were detected. Flow and laser scanning cytometry studies allowed us to differentiate three hemocyte populations, agranulocytes (R1) and granulocytes (R2 and R3). The exposure time to Hg increased the average red fluorescence (ARF) of agranulocytes and small granulocytes, while there was no change in large granulocytes, which showed a loss of membrane integrity. In control oysters, the three hemocyte populations showed an increase of ARF after 19 days of exposure although initial values were restored after 30 days. The average green fluorescence (AGF) was more stable than the ARF throughout the experiment. In Hg-exposed oysters, the values of AGF of agranulocytes showed an increase at half Hg-exposure period while the AGF values of large granulocytes decreased throughout the experiment, confirming the instability of these types of cells. The relative percentage of small granulocytes and granulocytes showed time variations in both control and exposed oysters. However, the values of small granulocytes remained constant during the whole experiment. The fact that there were only changes in agranulocytes and large granulocytes suggested a possible relationship between these two types of cells. In a quantitative study, we found a significant linear relationship between the agranulocytes and large granulocytes.

Animals↗
PubMed2002

A new cytometric method for the immunophenotypic characterization of bone-derived human osteoclasts.

BACKGROUND: Osteoclast cell function relates to bone resorption. Isolation and characterization of these cells from in vivo sources remain difficult. The aim of this study was to show the feasibility of using flow cytometry to identify and characterize human mature osteoclasts obtained from bone tissues. METHODS: Bone femoral heads obtained as discarded surgical material were used. To check the nature of 121F(+) (a monoclonal antibody specific for human osteoclasts) cells by flow cytometry, we used laser scanning cytometry to analyze simultaneously the immunophenotype and DNA cell content of osteoclast-like cell-enriched bone samples. RESULTS: Results were compared with conventional morphologic and cytochemical studies. The percentage of cells that showed both cytochemical (tartrate-resistant acid phosphatase [TRAP](+)) and immunophenotypic (121F(+)) osteoclast-associated characteristics was very similar (12.5 +/- 6.2 versus 14.7 +/- 11.7; P = 0.46). Laser scanning cytometry showed that 121F(+) cells were bigger (P = 0.04) and they had a higher DNA cell content (P = 0.04) and more nuclei per cell (P = 0.04) than the 121F(-) cells present in the same sample. DISCUSSION: This study relied on the combined use of the 121F(+) antibody and different cytometry-based techniques to characterize the osteoclast populations from human bone.

Acid Phosphatase↗
PubMed2004

Scanning fluorescent microscopy analysis is applicable for absolute and relative cell frequency determinations.

BACKGROUND: Flow cytometry (FCM) and laser scanning cytometry (LSC) are the routine techniques for fluorescent cell analysis. Recently, we developed a scanning fluorescent microscopy (SFM) technique. This study compares SFM to LSC (two slide-based cytometry, SBC, techniques) and FCM, in experimental and clinical settings. METHODS: For the relative cell-frequency determinations, HT29 colorectal cancer cells and Ficoll separated blood mononuclear cells (FSBMCs) were serially diluted (from 1:1 to 1:1,000) and measured by each of the three techniques. For the absolute cell number determinations (only for SBC) FSBMCs were smeared on slides, then HT29 cells were placed on the slide with a micromanipulator (5-50 cells). Tumor cells circulating in the peripheral blood were isolated by magnetic separation from clinical blood samples of colorectal cancer patients. All samples were double-stained by CD45 ECD and CAM5.2 FITC antibodies. For slides, TOTO-3 and Hoechst 33258 DNA dyes were applied as nuclear counter staining. RESULTS: In the relative cell frequency determinations, the correlations between the calculated value and measured values by SFM, LSC, and FCM were r(2) = 0.79, 0.62, and 0.84, respectively (for all P < 0.01). In the absolute cell frequency determinations, SFM and LSC correlated to a high degree (r(2) = 0.97; P < 0.01). CONCLUSIONS: SFM proved to be a reliable alternative method, providing results comparable to LSC and FCM. SBC proved to be more suitable for rare-cell detection than FCM. SFM with digital slides may prove an acceptable adaptation of conventional fluorescent microscopes in order to perform rare-cell detection.

Antiporters↗
PubMedDate not supplied

Prenatal diagnosis of a (X;X) translocation by fluorescence in situ hybridization and laser scanning image cytometry.

A de novo structural abnormality of one X chromosome was prenatally detected in a female fetus. This chromosomal abnormality has been analyzed by conventional cytogenetic methods, fluorescence in situ hybridization, and laser scanning image cytometry. The association of these techniques has demonstrated that this anomaly corresponds to a (X;X) translocation. Analysis of hybridization signals by laser scanning image cytometry allowed to localize that the breakpoints were at the X-centromeric region and Xp11.3, respectively. These results show the usefulness of image analysis and fluorescence in situ hybridization for a rapid characterization of de novo structural chromosome anomalies in prenatal diagnosis.

Adult↗