Phytoplankton group dynamics in the Bay of Marseilles during a 2-year survey based on analytical flow cytometry.

BACKGROUND: The Bay of Marseilles is under the influence of a large urban concentration and its maritime activities. All of them discharge compounds (hydrocarbons, excess nutrients, heavy metals, chemicals, etc.) that can alter the marine ecosystem. To investigate whether ultraphytoplankton (<10 microm) could be used as biosensors for their own ecosystem, a 2-year survey was conducted in the Bay of Marseilles. METHODS: Seven stations monitored water mass and potential anthropic effects in the bay. Seawater samples were collected monthly or bimonthly at three depths, prefiltered, fixed, and kept in liquid nitrogen until flow cytometric analysis. RESULTS: Five categories were created: Prochlorococcus, picoeukaryotes (<2 microm), nanoeukaryotes I (2--6 microm), nanoeukaryotes II (6--10 microm), and Synechococcus (<1.5 microm). Artificial neural network analysis (Kohonen self-organizing maps) produced the same number of clusters as cluster analysis with Winlist software (Verity Software House). CONCLUSIONS: In addition to the wide variabilities in abundance and biomass, there were a strong seasonal signal and sporadic events. Lessons are derived from this study for future monitoring of marine microorganisms.     

Flow cytometric assay for evaluation of the effects of cell density on cytotoxicity and induction of apoptosis

BACKGROUND: We used a flow cytometric assay, which allows us to perform precise measurements within a wide range of cell concentrations to study the effect of the density of cultured cells on their sensitivity to cytotoxic compounds. METHODS: To measure cytotoxic action, cells are plated in a 96-well plate at a density ranging from 700 to 100,000 cells/ml and are allowed to grow for 72 h in the presence of various concentrations of a cytotoxic agent. To quantitate the number of surviving cells, each sample is analyzed in a flow cytometer with equal acquisition time. Viable cells are identified by light scattering characteristics identical to those for untreated cells. To estimate the amount of viable, apoptotic, or necrotic (late apoptotic) cells, the samples are stained with Annexin V and propidium iodide. RESULTS: Using this method, we found that the cytotoxicity of ascorbic acid for malignant lymphoid CEM-C7 cells can be increased significantly when cell density decreases, reaching a value that is typically lower than the normal physiological concentration of ascorbic acid in blood. CONCLUSION: The flow cytometric analysis described in this study can be useful in comparing the effects of cell density on the cytotoxic action of various compounds.     

Flow cytometry controls, instrument setup, and the determination of positivity.

A frequent goal of flow cytometric analysis is to classify cells as positive or negative for a given marker, or to determine the precise ratio of positive to negative cells. This requires good and reproducible instrument setup, and careful use of controls for analyzing and interpreting the data. The type of controls to include in various kinds of flow cytometry experiments is a matter of some debate and discussion. In this tutorial, we classify controls in various categories, describe the options within each category, and discuss the merits of each option.     

Ultra- and microplankton assemblages as indicators of trophic status in a Mediterranean lagoon

The seasonal abundance distribution of heterotrophic prokaryotes, pico- and nanophytoplankton, was investigated in connection with environmental variables and microplankton abundance at five stations in Ghar El Melh Lagoon (northeastern Tunisia). Flow cytometry analysis of ultraplankton resolved (i) five heterotrophic prokaryote groups labelled LNA1, LNA2 (low nucleic acid content), HNA1, HNA2 and HNA3 (high nucleic acid content) and (ii) at least 14 ultraphytoplankton groups assigned to picoeukaryotes, picoprokaryotes, nanoeukaryotes, cryptophyte-like cells and some unknown communities. Redundancy analysis (RDA) revealed (i) autumn-summer outbreaks of heterotrophic prokaryotes dominated by HNA groups and (ii) winter-summer proliferation of ultraphytoplankton dominated by nanophytoplankton groups. Generalized additive models (GAM) highlighted the role of (i) water temperature and orthophosphate concentrations in heterotrophic prokaryote distribution and (ii) water temperature and salinity in ultraphytoplankton abundance variation. Based on Spearman's rank correlation, significant negative correlations were established between ultra- and microplankton communities suggesting that, through grazing pressure, microplankton may be behind the drastic decrease in ultraplankton abundances in spring.     

Flow cytometric DNA content analysis of neoplastic cells in haemolymph of the cockle Cerastoderma edule

Epizootiological outbreaks of disseminated neoplasia (DN) have been reported in association with mass mortalities in various bivalve species including the cockle Cerastoderma edule. A flow cytometric (FCM) procedure to study DNA content was successfully adapted and tested in haemolymph cells (haemocytes and neoplastic cells) of the cockle. The FCM results were similar to those obtained by histological analysis (DN diagnosis and haemolymph cell features). FCM analysis revealed differences in DNA content among normal haemocytes (diploid) and neoplastic cells. Four types of cells with abnormal DNA content were found in the haemolymph of affected animals: hypodiploid, hyperdiploid, triploid-sesploid and pentaploid. Our results suggest that the flow cytometric DNA content analysis can be applied to identify neoplastic cell types and to study the association between different cell types and the DN progression or remission in this edible and commercially important bivalve species.     

Intracellular HSP72 detection in HL60 cells using a flow cytometry system based on microfluidic analysis

HSP72 is an important marker for various environmental stresses and diseases, and many researchers need to detect HSP72 levels in various cells. We have therefore developed an assay to monitor intracellular heat-shock protein 72 expression on a microfluidic Lab-on-a-chip platform. We established this method to detect HSP72 intracellularly by antibody staining with DNA counterstaining. The Lab-on-a-chip technology is simple and efficient when performing flow cytometric assays. By permeabilizing the cells for the delivery of antibodies, we were able to show HSP72 expression after 30 min heat-shock at 44 degrees C and then at various post-incubation times at 37 degrees C. We compared our method to a conventional flow cytometer and an enzyme immunoassay technique.     

Quantifying Distribution of Flow Cytometric TCR-Vβ Usage with Economic Statistics

Measuring changes of the T cell receptor (TCR) repertoire is important to many fields of medicine. Flow cytometry is a popular technique to study the TCR repertoire, as it quickly provides insight into the TCR-Vβ usage among well-defined populations of T cells. However, the interpretation of the flow cytometric data remains difficult, and subtle TCR repertoire changes may go undetected. Here, we introduce a novel means for analyzing the flow cytometric data on TCR-Vβ usage. By applying economic statistics, we calculated the Gini-TCR skewing index from the flow cytometric TCR-Vβ analysis. The Gini-TCR skewing index, which is a direct measure of TCR-Vβ distribution among T cells, allowed us to track subtle changes of the TCR repertoire among distinct populations of T cells. Application of the Gini-TCR skewing index to the flow cytometric TCR-Vβ analysis will greatly help to gain better understanding of the TCR repertoire in health and disease.     

Effect of fixation temperature on flow cytometric measurement of intracellular antibody content of hybridomas during batch culture

In order to investigate the effect of fixation temperature on flow cytometric measurement of intracellular antibody content of hybridoma cells, cells in different growth stages during a batch culture were fixed and stored at 4 and -20 °C, respectively. Flow cytometric analysis indicates that both fixation temperatures can be used in monitoring the changes in intracellular antibody content of the cells during a batch culture. However, it is better to fix and store the cells at -20 °C than 4 °C with regard to preservation of intracellular antibody and storage stability.     

B cell activation. II. Receptor cross-linking by thymus-independent and thymus-dependent antigens induces a rapid decrease in the plasma membrane potential of antigen-binding B lymphocytes

We report the specific induction of B cell plasma membrane depolarization with the use of thymus-dependent and -independent antigens. We have utilized various trinitrophenol-carrier conjugates for the stimulation of isolated trinitrophenol-binding mouse B cells. Membrane depolarization was assessed by flow cytometric analysis of 3-3'-pentyloxacarbocyanine iodide (DiOC5[3])-stained cells. Entry into the cell cycle was determined by flow cytometric analysis of acridine orange-stained cells. The results indicate that polyvalent antigens, but not free hapten, induce B cell membrane depolarization by a large proportion of antigen-binding cells within 2 hr of stimulation. Although all polyvalent antigens induce membrane depolarization, only thymus-independent antigens induce the subsequent G0 to G1 transition, suggesting that the membrane Ig cross-linking signal alone, although sufficient to induce membrane depolarization and subsequent increased IA expression, is insufficient to drive the entry of B cells into the cell cycle. The G0 to G1 transition appears to be dependent on a second signal, perhaps mediated by the thymus-independent carrier or antigen-specific, Ia-restricted T cell helper.     

Differentiation of HL-60 promyelocytic leukemia cells monitored by flow cytometric measurement of nitro blue tetrazolium (NBT) reduction

Reduction of nitro blue tetrazolium (NBT) to insoluble blue formazan granules occurs during the stimulus-induced respiratory burst of mature granulocytes and is routinely used as an indicator of the extent of granulocytic differentiation of HL-60 acute promyelocytic leukemia cells. In the present study, the differentiation of HL-60 leukemia cells induced by dimethylsulfoxide (DMSO) or retinoic acid was monitored by flow cytometric (FCM) measurement of forward and 90 degree light scatter of NBT treated cells. Two-parameter correlated analysis permitted a distinction between cells with increased forward and decreased 90 degree light scatter (NBT-), and cells with decreased forward and increased 90 degree light scatter (NBT+). Fixation of NBT treated cells with 1% paraformaldehyde facilitated flow cytometric analysis, and allowed differences in NBT reduction to be quantitated. DMSO-induced cells expressed an all-or-none reduction of NBT to formazan, compared with retinoic acid treated cells that exhibited a graded response. Three parameter flow cytometric analysis of HL-60 leukemia cells stained with propidium iodide in combination with NBT allowed the determination of the cell cycle distribution of NBT-treated cells.     

Establish a flow cytometric method for quantitative detection of Beclin-1 expression

Flow cytometry is an advanced technology for efficient, rapid, specific and multi-parameter analysis of single cells in various basic research fields including cytobiology, immunology, genetic, hematology and other basic research. Beclin-1 protein is an important indicator in monitoring autophagic activity. However, quantitative flow cytometry had been rarely reported till now to be applied in the detection of Beclin-1 expression. The present study was aimed to establish a flow cytometric method for quantitative detection of Beclin-1 expression by employing the autophagy inhibitor 3-methyladenine as the control. A multi-parameter optimal method for Beclin-1 protein staining is as follows. 2 % bovine serum albumin in phosphate buffered saline was used for sample block. Concentration of primary antibody was 0.004 μg/μL. Samples were incubated at room temperature (25 °C) for 30 min. The prepared samples had better to be detected immediately or to be stored at 4 °C and detected within 6 h, otherwise the samples should be fixed in 1 % paraformaldehyde storing at 4 °C and detected within 3 d. Furthermore, we employed the immunohistochemistry to validate the method in vivo, the results confirmed flow cytometric method. The established flow cytometric analysis for Beclin-1 protein has the advantage of simpleness, speediness, sensitivity and reproducibility.     

Phytoplankton monitoring by high performance flow cytometry: a successful approach?

BACKGROUND: Regular phytoplankton monitoring in Dutch coastal waters is performed as an indicator of the ecological state of these waters. The monitoring program is focused on temporal and spatial changes of species composition and abundance. Flow cytometry has been introduced to provide additional information, to improve ecosystem understanding, and to increase the efficiency of analysis and reportage. METHODS: Phytoplankton community abundance and composition were routinely determined by flow cytometry and microscopy at six locations in the North Sea over three annual cycles between 2000 and 2003. Supplementary measurements were also made for fluorescence (chlorophyll-a and other pigments) and, in combination with flow cytometric and microscopic data, were used to determine phytoplankton abundance and composition as a function of their size distribution. Real-time imaging of species was also used to identify species on the basis of their flow cytometric optical characteristics. RESULTS: Flow cytometric analysis took 15 min on average. Analysis including data processing, and Web site reportage took less than 1 h. Phytoplankton concentrations (cells/ml), biomass (fluorescence/ml), and concentration of phycoerythrin- or phycocyanin-containing cells (cells/ml) as a function of their algal size were produced every 2 weeks on average. The phytoplankton integrated annual concentration and biomass were used as ecological indicators for overall phytoplankton status. Real-time imaging of cells in flow enabled the identification of dominant species and was applied as an early warning system for Phaeocystis spp. CONCLUSIONS: The reproducibility and count precision due to the large number of observations of the flow cytometric technique provided reliable data for monitoring long-term trends. Flow cytometrically based analyses extended the lower detection limit (<0.5 microm) of analysis beyond the capabilities of other techniques such as the relation between small and larger phytoplankton, the relation between cell counts and biomass as a function of cell size, but also the ability to monitor and report on blooms of harmful algae. A good correlation was found between concentrations (cells/ml) measured by flow cytometry and microscopy. In practice, flow cytometric analysis of a single marine sample took 15 min on average.     

Bivariate flow cytometric analysis of murine intestinal epithelial cells for cytokinetic studies

The heterogeneous nature of the small intestine and the lack of methods to obtain pure crypt populations has, in the past, limited the application of standard flow cytometric analysis for cytokinetic studies of the proliferating crypts. We describe a flow cytometric technique to discriminate crypt and villus cells in an epithelial cell suspension on the basis of cell length, and to measure the DNA content of the discriminated subpopulations. Our data indicate that bivariate analysis of a mixed epithelial cell suspension can be used to distinguish mature villus cells, G1 crypt cells, and S-phase crypt cells. In addition, continuous labeling studies suggest that the position of a cell on the cell length axis reflects epithelial cell maturity. We applied this flow cytometric technique to determine the cytokinetic nature of epithelial cells obtained by sequential digestion of the small intestine.     

The flow cytometric PKH-26 assay for the determination of T-cell mediated cytotoxic activity

We present a rapid flow cytometric and non-radioactive functional assay developed for the determination of the cytotoxic activity of T lymphocytes, natural killer cells, and lymphokine-activated killer cells. In contrast to indirect evaluation of cytotoxicity using radioactive assays, this assay is based on the quantitative and qualitative flow cytometric analysis of cell damage on a single cell level. Target cells are stained with PKH-26, a lipophilic dye that stably integrates into the cell membrane, without disturbing its surface marker expression. It, thus, permits the distinction between target and effector cells. After short term in vitro incubation (1.5-3h), AnnexinV-FITC (ann-FITC) staining allows to discriminate between apoptotic and non-apoptotic target cells. Data analysis is performed first by gating on PKH-26 positive target cells, followed by the analysis of the ann-FITC positive subpopulation. The percentage of cytotoxicity in the PKH-26 gated cell population is calculated by subtracting unspecific ann-FITC positive target cells, measured in appropriate controls without effector cells. Using in vitro generated antigen-specific cytotoxic T lymphocytes, we demonstrate that this flow cytometric assay is sensitive, correlates well with the standard 51Cr release assay, and is easy to handle.     

Fluorescence kinetics in HeLa cells after treatment with cell cycle arrest inducers visualized with Fucci (fluorescent ubiquitination-based cell cycle indicator)

Fucci (fluorescent ubiquitination-based cell cycle indicator) is able to visualize dynamics of cell cycle progression in live cells; G1- and S-/G2-/M-phase cells expressing Fucci emit red and green fluorescence, respectively. This system could be applied to cell kinetic analysis of tumour cells in the field of cancer therapy; however, it is still unclear how fluorescence kinetics change after various treatments, including exposure to anticancer agents. To explore this, we arrested live HeLa cells expressing the Fucci probes at various cell cycle stages and observed the fluorescence, in conjunction with flow cytometric analysis. X-irradiation, HU (hydroxyurea) and nocodazole arrest cells at G2/M boundary, early S-phase and early M-phase, respectively. Although X-irradiation and HU treatment induced similar accumulation kinetics of green fluorescent cells, nocodazole treatment induced an abnormal red fluorescence at M phase, followed by accumulation of both red and green fluorescent cells with 4N DNA content. We conclude that certain agents that disrupt normal cell cycle regulation could cause unexpected fluorescence kinetics in the Fucci system.     

A single-cell assay of beta-galactosidase in recombinant Escherichia coli using flow cytometry

A flow cytometric method was developed for the assay of beta-galactosidase in single Escherichia coli cells. A new fluorogenic substrate for beta-galactosidase, C(12)FDG, contains a lipophilic group that allows the substrate to penetrate through cell membranes under normal conditions. When the substrate is hydrolyzed by intracellular beta-galactosidase, a green fluorescent product is formed and retained inside the cell. Consequently, the stained beta-galactosidase-positive cells exhibit fluorescence, which is detected by flow cytometry. This new assay was used to analyze the segregational instability caused by a reduction in specific growth rate of the plasmid-bearing cells in the T7 expression system. Induction results in a substantial accumulation of intracellular beta-galactosidase along with a rapid increase in the fraction of plasmid-free cells. Once the cells lose the plasmid, they no longer produce beta-galactosidase, which is reduced by at least half every generation; thus, after staining, the fluorescent, plasmid-bearing cells can be distinguished from the nonfluorescent, plasmid-free cells using flow cytometry. This article describes the feasibility of the flow cytometric assay for single E. coli cells and reports the optimal assay conditions. A direct relationship between beta-galactosidase activity and green fluorescence intensity was found, and the fractions of recombinant cells in batch cultures were analyzed after various levels of induction.     

The presence of a side population and its marker ABCG2 in human deciduous dental pulp cells

Stem cells have been identified using the DNA-binding dye Hoechst 33342 and flow cytometry (FCM) in various tissues known as the side population (SP). The present study shows, for the first time, the presence of side population cells in human deciduous dental pulp cells (DPCs). Flow cytometric identification revealed that 2% of human deciduous DPCs were SP cells and that this SP profile disappeared in the presence of verapamil. The SP marker ABCG2 protein was localized to DPCs in the cell membrane by immunofluorescence staining, and flow cytometric analysis demonstrated that 3.6% of DPCs were ABCG2-positive. Furthermore, quantitative real-time PCR proved that ABCG2 mRNA expression in DPCs isolated from human exfoliated deciduous teeth was higher than in DPCs from permanent teeth. Our findings demonstrate that DPCs from human exfoliated deciduous teeth contain a higher proportion of the SP phenotype than permanent teeth and that they may constitute a stem cell population.     

Cytomat-R: a computer-controlled multiple laser source multiparameter flow cytophotometer system.

A multiple illumination wavelength multiparameter flow cytophotometer system, using laser sources and controlled by a small, general-purpose digital computer, has been produced for use in the development of new flow cytometric techniques. Three different laser wave-lengths can be used simultaneously to illuminate different regions of the flow chamber; as many as five measurements of light scattering at various angles, extinction, and fluorescence at one or more wavelengths can be made at each illuminated station. Cells in suspension may be examined at rates of 1000 cells/sec, with seven correlated optical measurements being recorded for each cell. A library of programs for data manipulation and statistical analysis make it possible to use the system to develop and implement cell characterization, counting and classification procedures for basic and clinical research applications.