64 research outputs found
Analysis of Nucleocytoplasmic Protein Shuttling by Imaging Flow Cytometry
No full textMany intracellular signal transduction events involve the reversible shuttling of proteins between the cytoplasm and the nucleus. Study of these processes requires imaging information on the protein localization in a given cell and a large number of measurements to obtain sufficient statistics on the protein localization in the whole population. The protocol describes method for quantitative imaging flow cytometry analysis of intracellular distribution of NF-kappaB in ARPE-19 cells stained with specific fluorochrome-conjugated antibodies. The described technique alone or in combination with standard flow cytometry methods can be applied to study any protein undergoing translocation from cytoplasm into the nucleus in a variety of cell lines as well as in heterogeneous primary cell population
Imaging Flow Cytometry of Legionella-Containing Vacuoles in Intact and Homogenized Wild-Type and Mutant Dictyostelium
No full textThe causative agent of a severe pneumonia termed "Legionnaires' disease", Legionella pneumophila, replicates within protozoan and mammalian phagocytes in a specialized intracellular compartment called the Legionella-containing vacuole (LCV). This compartment does not fuse with bactericidal lysosomes but communicates extensively with several cellular vesicle trafficking pathways and eventually associates tightly with the endoplasmic reticulum. In order to comprehend in detail the complex process of LCV formation, the identification and kinetic analysis of cellular trafficking pathway markers on the pathogen vacuole are crucial. This chapter describes imaging flow cytometry (IFC)-based methods for the objective, quantitative and high-throughput analysis of different fluorescently tagged proteins or probes on the LCV. To this end, we use the haploid amoeba Dictyostelium discoideum as an infection model for L. pneumophila, to analyze either fixed intact infected host cells or LCVs from homogenized amoebae. Parental strains and isogenic mutant amoebae are compared in order to determine the contribution of a specific host factor to LCV formation. The amoebae simultaneously produce two different fluorescently tagged probes enabling tandem quantification of two LCV markers in intact amoebae or the identification of LCVs using one probe and quantification of the other probe in host cell homogenates. The IFC approach allows rapid generation of statistically robust data from thousands of pathogen vacuoles and can be applied to other infection models
Lysine dendrimers and starburst copolymers of lysine and glutamic acid as carriers of anticancer drugs based on the complexes of platinum and gold
No full textMulti-parametric imaging of cell heterogeneity in apoptosis analysis
No full textAbstract Apoptosis is a multistep process of programmed cell death where different morphological and molecular events occur simultaneously and/or consequently. Recent progress in programmed cell death analysis uncovered large heterogeneity in response of individual cells to the apoptotic stimuli. Analysis of the complex and dynamic process of apoptosis requires a capacity to quantitate multiparametric data obtained from multicolor labeling and/or fluorescent reporters of live cells in conjunction with morphological analysis. Modern methods of multiparametric apoptosis study include but are not limited to fluorescent microscopy, flow cytometry and imaging flow cytometry.In the current review we discuss the image-based evaluation of apoptosis on the single-cell and population level by imaging flow cytometry in parallel with other techniques. The advantage of imaging flow cytometry is its ability to interrogate multiparametric morphometric and fluorescence quantitative data in statistically robust manner. Here we describe the current status and future perspectives of this emerging field, as well as some challenges and limitations. We also highlight a number of assays and multicolor labeling probes, utilizing both microscopy and different variants of imaging cytometry, including commonly based assays and novel developments in the field
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Microvesicles and intercellular communication in the context of parasitism
No full textThere is a rapidly growing body of evidence that production of microvesicles (MVs) is a universal feature of cellular life. MVs can incorporate microRNA (miRNA), mRNA, mtDNA, DNA and retrotransposons, camouflage viruses/viral components from immune surveillance, and transfer cargo between cells. These properties make MVs an essential player in intercellular communication. Increasing evidence supports the notion that MVs can also act as long-distance vehicles for RNA molecules and participate in metabolic synchronization and reprogramming eukaryotic cells including stem and germinal cells. MV ability to carry on DNA and their general distribution makes them attractive candidates for horizontal gene transfer, particularly between multi-cellular organisms and their parasites; this suggests important implications for the co-evolution of parasites and their hosts. In this review, we provide current understanding of the roles played by MVs in intracellular pathogens and parasitic infections. We also discuss the possible role of MVs in co-infection and host shifting.Version of Recor
Diazepam inhibits cell respiration and induces fragmentation of mitochondrial reticulum
No full textAbstractDiazepam (70–150 μg/ml) significantly inhibits oxygen consumption by pig kidney embryo cells and causes the cellular ATP level to fall. The maximum inhibitory effect develops after 1.5–2.5 h of diazepam treatment. In isolated mitochondria diazepam inhibits respiration in state 2 and 3u with glutamate and in state 3u with succinate. Ethylrhodamine staining and electron microscopic study reveal fragmentation of mitochondria in living cells
Dynamic microtubules drive fibroblast spreading
No full textWhen cells with a mesenchymal type of motility come into contact with an adhesive substrate they adhere and start spreading by the formation of lamellipodia. Using a label-free approach and virtual synchronization approach we analyzed spreading in fibroblasts and cancer cells. In all cell lines spreading is a non-linear process undergoing isotropic or anisotropic modes with first fast (5–20 min) and then slow (30–120 min) phases. In the first 10 min cell area increases 2–4 times, while the absolute rate of initial spreading decreases 2–8 times. Fast spreading depends on actin polymerization and dynamic microtubules. Inhibition of microtubule growth was sufficient for a slowdown of initial spreading. Inhibition of myosin II in the presence of stable microtubules restored fast spreading. Inhibition of actin polymerization or complete depolymerization of microtubules slowed down fast spreading. However, in these cases inhibition of myosin II only partially restored spreading kinetics. We conclude that rapid growth of microtubules towards cell margins at the first stage of cell spreading temporarily inhibits phosphorylation of myosin II and is essential for the fast isotropic spreading. Comparison of the fibroblasts with cancer cells shows that fast spreading in different cell types shares similar kinetics and mechanisms, and strongly depends on dynamic microtubules
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