Using flow cytometry for in situ monitoring of antimicrobial compound production in the biocontrol bacterium Pseudomonas fluorescens CHA0

Pseudomonas fluorescens strain CHA0 is able to protect plants against a variety of pathogens, notably by producing the two antimicrobial compounds 2,4-diacetylphloroglucinol (DAPG) and pyoluteorin (PLT). The regulation of the expression of these compounds is affected by many biotic factors, such as fungal pathogens, rhizosphere bacteria as well as plant species. Therefore, the influence of some plant phenolic compounds on the expression of DAPG and PLT biosynthetic genes has been tested using GFP-based reporter, monitored by standard fluometry and flow cytometry. In situ experiments were also performed with cucumber plants. We found that several plant metabolites such as IAA and umbelliferone are able to modify significantly the expression of DAPG and PLT. The use of flow cytometry with autofluorescents proteins seems to be a promising method to study rhizobacteria-plant interactions.

Detection of plant-modulated alterations in antifungal gene expression in Pseudomonas fluorescens CHA0 on roots by flow cytometry.

The biocontrol activity of the root-colonizing Pseudomonas fluorescens strain CHA0 is largely determined by the production of antifungal metabolites, especially 2,4-diacetylphloroglucinol. The expression of these metabolites depends on abiotic and biotic environmental factors, in particular, elements present in the rhizosphere. In this study, we have developed a new method for the in situ analysis of antifungal gene expression using flow cytometry combined with green fluorescent protein (GFP)-based reporter fusions to the phlA and prnA genes essential for the production of the antifungal compounds 2,4-diacetylphloroglucinol and pyrrolnitrin, respectively, in strain CHA0. Expression of phlA-gfp and prnA-gfp in CHA0 cells harvested from the rhizosphere of a set of plant species as well as from the roots of healthy, leaf pathogen-attacked, and physically stressed plants were analyzed using a FACSCalibur. After subtraction of background fluorescence emitted by plant-derived particles and CHA0 cells not carrying the gfp reporters, the average gene expression per bacterial cell could be calculated. Levels of phlA and prnA expression varied significantly in the rhizospheres of different plant species. Physical stress and leaf pathogen infection lowered phlA expression levels in the rhizosphere of cucumber. Our results demonstrate that the newly developed approach is suitable to monitor differences in levels of antifungal gene expression in response to various plant-derived factors. An advantage of the method is that it allows quantification of bacterial gene expression in rhizosphere populations at a single-cell level. To our best knowledge, this is the first study using flow cytometry for the in situ analysis of biocontrol gene expression in a plant-beneficial bacterium in the rhizosphere.

LiveCount Assay: concomitant measurement of cytolytic activity and phenotypic characterisation of CD8(+) T-cells by flow cytometry.

Tumour immunologists strive to develop efficient tumour vaccination and adoptive transfer therapies that enlarge the pool of tumour-specific and -reactive effector T-cells in vivo. To assess the efficiency of the various strategies, ex vivo assays are needed for the longitudinal monitoring of the patient's specific immune responses providing both quantitative and qualitative data. In particular, since tumour cell cytolysis is the end goal of tumour immunotherapy, routine immune monitoring protocols need to include a read-out for the cytolytic efficiency of Ag-specific cells. We propose to combine current immune monitoring techniques in a highly sensitive and reproducible multi-parametric flow cytometry based cytotoxicity assay that has been optimised to require low numbers of Ag-specific T-cells. The possibility of re-analysing those T-cells that have undergone lytic activity is illustrated by the concomitant detection of CD107a upregulation on the surface of degranulated T-cells. To date, the LiveCount Assay provides the only possibility of assessing the ex vivo cytolytic activity of low-frequency Ag-specific cytotoxic T-lymphocytes from patient material.

Flow cytometry in the analysis of cellular populations

Flow cytometry has become a valuable tool in cell biology. By analyzing large number of cells individually using light-scatter and fluorescence measurements, this technique reveals both cellular characteristics and the levels of cellular components. Flow cytometry has been developed to rapidly enumerate cells and to distinguish among different cell stages and structures using multiple staining. In addition to high-speed multiparametric data acquisition, analysis and cell sorting, which allow other characteristics of individual cells to be studied, have increased the interest of researchers in this technique. This chapter gives an overview of the principles of flow cytometry and examples of the application ofthe technique.

Use of phycoerythrin and allophycocyanin for fluorescence resonance energy transfer analyzed by flow cytometry: advantages and limitations.

BACKGROUND: This study validates the use of phycoerythrin (PE) and allophycocyanin (APC) for fluorescence energy transfer (FRET) analyzed by flow cytometry. METHODS: FRET was detected when a pair of antibody conjugates directed against two noncompetitive epitopes on the same CD8alpha chain was used. FRET was also detected between antibody conjugate pairs specific for the two chains of the heterodimeric alpha (4)beta(1) integrin. Similarly, the association of T-cell receptor (TCR) with a soluble antigen ligand was detected by FRET when anti-TCR antibody and MHC class I/peptide complexes (<<tetramers>>) were used. RESULTS: FRET efficiency was always less than 10%, probably because of steric effects associated with the size and structure of PE and APC. Some suggestions are given to take into account this and other effects (e.g., donor and acceptor concentrations) for a better interpretation of FRET results obtained with this pair of fluorochromes. CONCLUSIONS: We conclude that FRET assays can be carried out easily with commercially available antibodies and flow cytometers to study arrays of multimolecular complexes.

Toxicity of triclosan, penconazole and metalaxyl on Caulobacter crescentus and a freshwater microbial community as assessed by flow cytometry.

Biocides are widely used for domestic hygiene, agricultural and industrial applications. Their widespread use has resulted in their introduction into the environment and raised concerns about potential deleterious effects on aquatic ecosystems. In this study, the toxicity of the biocides triclosan, penconazole and metalaxyl were evaluated with the freshwater bacterium Caulobacter crescentus and with a freshwater microbial community using a combination of single- and double-stain flow cytometric assays. Growth of C.  crescentus and the freshwater community were repressed by triclosan but not by penconazole or metalaxyl at concentrations up to 250 μM. The repressive effect of triclosan was dependent on culture conditions. Caulobacter crescentus was more sensitive to triclosan when grown with high glucose at high cell density than when grown directly in sterilized lake water at low cell density. This suggests that the use of conventional growth conditions may overestimate biocide toxicity. Additional experiments showed that the freshwater community was more sensitive to triclosan than C.  crescentus, with 10 nM of triclosan being sufficient to repress growth and change the phylogenetic composition of the community. These results demonstrate that isolate-based assays may underestimate biocide toxicity and highlight the importance of assessing toxicity directly on natural microbial communities. Because 10 nM of triclosan is within the range of concentrations observed in freshwater systems, these results also raise concerns about the risk of introducing triclosan into the environment.

Using digital RNA counting and flow cytometry to compare mRNA with protein expression in acute leukemias.

BACKGROUND: The diagnosis of malignant hematologic diseases has become increasingly complex during the last decade. It is based on the interpretation of results from different laboratory analyses, which range from microscopy to gene expression profiling. Recently, a method for the analysis of RNA phenotypes has been developed, the nCounter technology (Nanostring® Technologies), which allows for simultaneous quantification of hundreds of RNA molecules in biological samples. We evaluated this technique in a Swiss multi-center study on eighty-six samples from acute leukemia patients. METHODS: mRNA and protein profiles were established for normal peripheral blood and bone marrow samples. Signal intensities of the various tested antigens with surface expression were similar to those found in previously performed Affymetrix microarray analyses. Acute leukemia samples were analyzed for a set of twenty-two validated antigens and the Pearson Correlation Coefficient for nCounter and flow cytometry results was calculated. RESULTS: Highly significant values between 0.40 and 0.97 were found for the twenty-two antigens tested. A second correlation analysis performed on a per sample basis resulted in concordant results between flow cytometry and nCounter in 44-100% of the antigens tested (mean = 76%), depending on the number of blasts present in a sample, the homogeneity of the blast population, and the type of leukemia (AML or ALL). CONCLUSIONS: The nCounter technology allows for fast and easy depiction of a mRNA profile from hematologic samples. This technology has the potential to become a valuable tool for the diagnosis of acute leukemias, in addition to multi-color flow cytometry.

The use of fluorescent probes to assess viability of the plant pathogenic bacterium Clavibacter michiganensis subsp. michiganensis by flow cytometry

Determination of the viability of bacteria by the conventional plating technique is a time-consuming process. Methods based on enzyme activity or membrane integrity are much faster and may be good alternatives. Assessment of the viability of suspensions of the plant pathogenic bacterium Clavibacter michiganensis subsp. michiganensis (Cmm) using the fluorescent probes Calcein acetoxy methyl ester (Calcein AM), carboxyfluorescein diacetate (cFDA), and propidium iodide (PI) in combination with flow cytometry was evaluated. Heat-treated and viable (non-treated) Cmm cells labeled with Calcein AM, cFDA, PI, or combinations of Calcein AM and cFDA with PI, could be distinguished based on their fluorescence intensity in flow cytometry analysis. Non-treated cells showed relatively high green fluorescence levels due to staining with either Calcein AM or cFDA, whereas damaged cells (heat-treated) showed high red fluorescence levels due to staining with PI. Flow cytometry also allowed a rapid quantification of viable Cmm cells labeled with Calcein AM or cFDA and heat-treated cells labeled with PI. Therefore, the application of flow cytometry in combination with fluorescent probes appears to be a promising technique for assessing viability of Cmm cells when cells are labeled with Calcein AM or the combination of Calcein AM with PI.

Trypan blue exclusion assay by flow cytometry

Dye exclusion tests are used to determine the number of live and dead cells. These assays are based on the principle that intact plasma membranes in live cells exclude specific dyes, whereas dead cells do not. Although widely used, the trypan blue (TB) exclusion assay has limitations. The dye can be incorporated by live cells after a short exposure time, and personal reliability, related to the expertise of the analyst, can affect the results. We propose an alternative assay for evaluating cell viability that combines the TB exclusion test and the high sensitivity of the flow cytometry technique. Previous studies have demonstrated the ability of TB to emit fluorescence when complexed with proteins. According to our results, TB/bovine serum albumin and TB/cytoplasmic protein complexes emit fluorescence at 660 nm, which is detectable by flow cytometry using a 650-nm low-pass band filter. TB at 0.002% (w/v) was defined as the optimum concentration for distinguishing unstained living cells from fluorescent dead cells, and fluorescence emission was stable for 30 min after cell treatment. Although previous studies have shown that TB promotes green fluorescence quenching, TB at 0.002% did not interfere with green fluorescence in human live T-cells stained with anti-CD3/fluorescein isothiocyanate (FITC) monoclonal antibody. We observed a high correlation between the percentage of propidium iodide+CD3/FITC+ and TB+CD3/FITC+ cells, as well as similar double-stained cell profiles in flow cytometry dot-plot graphs. Taken together, the results indicate that a TB exclusion assay by flow cytometry can be employed as an alternative tool for quick and reliable cell viability analysis.

Analysis of testicular cell populations using flow cytometry

Studying testis is complex, because the tissue has a very heterogeneous cell composition and its structure changes dynamically during development. In reproductive field, the cell composition is traditionally studied by morphometric methods such as immunohistochemistry and immunofluorescence. These techniques provide accurate quantitative information about cell composition, cell-cell association and localization of the cells of interest. However, the sample preparation, processing, staining and data analysis are laborious and may take several working days. Flow cytometry protocols coupled with DNA stains have played an important role in providing quantitative information of testicular cells populations ex vivo and in vitro studies. Nevertheless, the addition of specific cells markers such as intracellular antibodies would allow the more specific identification of cells of crucial interest during spermatogenesis. For this study, adult rat Sprague-Dawley rats were used for optimization of the flow cytometry protocol. Specific steps within the protocol were optimized to obtain a singlecell suspension representative of the cell composition of the starting material. Fixation and permeabilization procedure were optimized to be compatible with DNA stains and fluorescent intracellular antibodies. Optimization was achieved by quantitative analysis of specific parameters such as recovery of meiotic cells, amount of debris and comparison of the proportions of the various cell populations with already published data. As a result, a new and fast flow cytometry method coupled with DNA stain and intracellular antigen detection was developed. This new technique is suitable for analysis of population behavior and specific cells during postnatal testis development and spermatogenesis in rodents. This rapid protocol recapitulated the known vimentin and γH2AX protein expression patterns during rodent testis ontogenesis. Moreover, the assay was applicable for phenotype characterization of SCRbKO and E2F1KO mouse models.

Standardization of cytokine flow cytometry assays

Affiliation: Faculté de médecine, Université de Montréal & CANVAC

Identification of haploid maize by flow cytometry, morphological and molecular markers

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

A flow cytometry method for testing the susceptibility of Cryptococcus spp. to amphotericin B

Human fungal infections have increased at an alarming rate in recent years, particularly in immunocompromised individuals. Cryptococcosis is the second most prevalent systemic fungal infection worldwide, and the most prevalent systemic infection in immunocompromised individuals, representing more than 70% of cases. The incidence of cryptococcosis is high in people with HIV/acquired immunodeficiency syndrome (AIDS), with recent estimates indicating that there are one million cases of cryptococcal meningitis globally per year in AIDS patients. The aim of this research was to develop a rapid flow cytometric antifungal susceptibility test and to compare the results with the standard methods. A reference strain and clinical isolates of Cryptococcus neoformans and Cryptococcus gattii were tested for susceptibility to amphotericin B by flow cytometry using propidium iodide as indicator of viability. Flow cytometry (FC) results were compared with the minimum inhibitory concentration (MIC) values determined by microdilution. The antifungal activity of amphotericin B ranged from MICs of 0.06 to 2μg/ml for the 11 isolates studied. The same results were found by FC. The FC method allows same-day results, assisting in the selection of appropriate antifungal therapies. These results demonstrate an excellent correlation between FC and the classic methods of testing for susceptibility to antifungal agents. This rapid diagnosis method makes it possible to quickly administer effective therapeutic interventions, often saving lives.