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Analyzing Chemotaxis Using Dictyostelium discoideum as a Model System

Dictyostelium discoideum are simple eukaryotic amoebas that when starved of a food source have the remarkable ability to spontaneously aggregate and differentiate to form a cluster of spores on top of a stalk of vacuolated cells (see Fig. 1 and ref. 1). The process of aggregation, although quite f ...

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Reconstructing Leukocyte Migration in 3D Extracellular Matrix by Time-Lapse Videomicroscopy and Computer-Assisted Tracking

The quantification of cell migration within three-dimensional (3D) extracellular matrix has become an important tool to investigate how cells integrate molecular events into higher-order adhesion and positioning within the tissue. Because most processes in vivo occur within 3D ...

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Evaluation of Rho Family Small G-Protein Activity Induced by Integrin Ligation on Human Leukocytes

Integrin engagement by natural ligands or specific antibodies (Abs) initiates a series of intracellular signaling events that are important for regulating different cell functions, including migration, adhesion, proliferation, differentiation, apoptosis, and specif ...

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Measurement of the Levels of Polymerized Actin (F-Actin) in Chemokine-Stimulated Lymphocytes and GFP-Coupled cDNA Transfected Lymphoid Cells by Flow C

The development of both microscopic and flow cytometry techniques has allowed investigators to examine the individual behavior of migratory cells under many different conditions, such as inflammation, invasion and metastasis, normal recirculation of blood cells, and so forth. C ...

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Chemotactic Profiling of Lymphocyte Subpopulations

Among a variety of trafficking signals, chemokines and their receptors represent key regulators of lymphocyte trafficking and localization under homeostatic as well as inflammatory conditions (1,2). Novel subsets of lymphocytes have been identified on the basis of their profile of ...

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Posttranslational Processing of Chemokines

Under physiological and pathological conditions, low-molecular-mass chemo-tactic cytokines or chemokines provide a chemotactic gradient for the directional migration of specific leukocyte subclasses. In vivo, leukocyte migration requires a network of molecules, inc ...

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The Expression of Exogenous Genes in Macrophages: Obstacles and Opportunities

Over the past three decades many techniques for expressing exogenous genes in a variety of cells and cell lines have been developed. Exogenous gene expression in macrophages has lagged behind that of other nonhematopioetic cells. There are many reasons for this, but most are due to technical di ...

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Exploiting Fc Chimaeric Proteins for the Identification of Ligands Specific for the Mannose Receptor

The use of chimaeric molecules bearing tags easily recognised by secondary reagents has facilitated the discovery of protein–protein and protein–carbohydrate interactions using binding assays in situ, in solution and in solid phase. In this chapter we describe our experience in the u ...

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Multivalent Protein Probes for the Identification and Characterization of Cognate Cellular Ligands for Myeloid Cell Surface Receptors

Cell–cell interactions mediated by cell surface receptor–ligand pairs in the immune system are often of low affinity and transient in nature. To begin to study these weak interactions, it is desirable to devise a generally applicable method for screening for and enriching cells expressing ...

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Probing In Vivo Origins of Mononuclear Phagocytes by Conditional Ablation and Reconstitution

Dendritic cells (DCs) and macrophages (MΦs), collectively termed mononuclear phagocytes (MP), are crucial for homeostatic tissue maintenance as well as the innate and adaptive host defense. These pleiotropic functions are likely to be covered by distinct DC and MΦ subsets, defined by an ...

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Detection of Activated Rab7 GTPase with an Immobilized RILP Probe

The dynamic and coordinated exchange of multiple GTPases between the cytosol and the phagosome membrane represents a critical process during phagosome biogenesis. In particular, acquisition of Rab7 is crucial for progression to the stage where formation of phagolysosomes is obse ...

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Analysis of Macrophage Phagocytosis: Quantitative Assays of Phagosome Formation and Maturation Using High-Throughput Fluorescence Microscopy

Phagocytosis of invading pathogens by macrophages represents a fundamental component of the innate immune system. In this chapter, we describe protocols designed for high-throughput analysis of phagosome formation and maturation using latex beads as model phagocytic targets. ...

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Macrophage Activation: Classical Versus Alternative

Macrophages are involved in both innate and adaptative immune responses. Depending on the types of cytokines that macrophages are exposed to, these cells are subjected to classical (Th1) or alternative (Th2) activation. In the first case, macrophages, particularly when activated by in ...

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Generation of Murine Growth Factor-Dependent Long-Term Dendritic Cell Lines to Investigate Host-Parasite Interactions

Substantial progress has been made over the last several years in the development of protocols for the isolation of large numbers of dendritic cells (DCs) from different tissues and their short-term culture. Indeed, several stable DC lines and clones have been established from various tiss ...

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Coordinate Regulation of Sphingosine Kinase and Actin Dynamics

The activation of lipid-modifying enzymes generally involves a physical change in their interactions with the membrane substrate. For sphingosine kinase, a predominantly cytosolic enzyme in resting cells, activation is accompanied by translocation to specific subsets of cel ...

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Large Scale Phagosome Preparation

Phagocytosis is the process by which cells engulf and destroy large particles such as pathogens or apoptotic cells. In this way, macrophages play a pivotal role in the resolution of microbial infections. However, many microorganisms have evolved efficient strategies to preempt the weap ...

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Generation of Retroviral Macrophage cDNA Expression Libraries and Functional Screening for Surface Receptors

Macrophages, tissue-based phagocytic cells derived from blood monocytes, play important roles in immunity and homeostasis. As professional scavengers, macrophages phagocytose microbes, apoptotic and necrotic cells and take up modified lipoprotein particles. However, m ...

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Use of Fluorescent Probes to Detect Lipid Signaling Intermediates in Macrophages

To fulfill their function in host defense, professional phagocytes such as neutrophils and macrophages exhibit the ability to ingest (phagocytose), kill, and dispose of pathogenic microorganisms. Recent studies have provided strong evidence for the importance of membrane lipi ...

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Stable Lentiviral Vector-Mediated Gene Silencing in Human Monocytic Cell Lines

A major obstacle in studying monocyte cell biology has been the resistance of these cells to genetic manipulation, particularly when using nonviral methods. In the approach outlined in this chapter, we describe a strategy for stable gene silencing of monocytic cells. Using a vesicular stom ...

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Identification of Pathogen Receptors on Dendritic Cells to Understand their Function and to Identify New Drug Targets

Dendritic cells (DCs) are crucial in the defence against invading pathogens. These professional antigen-presenting cells express a diversity of pattern recognition receptors to recognize pathogens and to induce adaptive immune responses. However, pathogens have also devel ...

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