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Modified Microchemotaxis Assays

Chemotaxis is the process by which leukocytes are directed to sites of inflammation under the influence of a concentration gradient of the soluble chemotactic molecules. Upon encountering a chemotactic molecule, responding leukocytes begin to migrate directionally from regio ...

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Generation of Stable Cell Lines Expressing Chemokine Receptors

Most human and murine leukocytes express multiple chemokine receptors with overlapping ligand specificity. Therefore the creation of stable cell lines expressing specific chemokine receptors greatly facilitates chemokine receptor characterization, particularly ...

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Transwell Chemotaxis

Numerous and diverse biological functions are regulated by chemokines. In addition to the well characterized proinflammatory activities such as integrin activation, chemotaxis, lipid mediator biosynthesis, superoxide radical production, and granule enzyme release (r ...

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Endothelial Cell Chemotaxis Assays

Endothelial cell migration and proliferation play an important role in the growth and development of new blood vessels, in the process known as neovascularization or angiogenesis. In the normal developing embryo, new blood vessels are formed by the directed movement of endothelial cel ...

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Radiolabeled Chemokine Binding Assays

Knowledge of a receptor’ pharmacology provides information which is often essential to unraveling the biological function of that receptor. The chemokine system represents a striking example as most receptors in the family bind multiple chemokines, often with complex overlappi ...

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Calcium Mobilization

The intracellular ionic composition is closely related to cellular activity. The key roles in controlling cell metabolism are played by calcium, hydrogen, sodium, and potassium concentrations. It is generally recognized that cells spend a great amount of their energy on stringent reg ...

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Actin Polymerization

Locomotion of leukocytes is a complex event requiring coordinated activity of cytoskeletal, membrane, and adhesion systems (1). Prior to the migration of cells into the inflamed tissue, they have to pass the endothelium. This process involves several types of adhesion molecule interac ...

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Histamine Release

Basophils and mast cells play an important role in allergic disorders. Both of these cell types produce and secrete many mediators that play an obligatory role in immediate hypersensitivity reactions. Some of the mediators are preformed and stored in granules. An example is histamine. Other ...

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Reactive Oxygen Release

Monocytes, macrophages, neutrophils, and eosinophils are able to generate and release reactive oxygen species. The reactive oxygen species are generated by reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase which is activated by a number of different soluble ...

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Measurement of Phosphoinositide 3-Kinase Activity

The term “PI 3-kinase” is now applied to a growing family of proteins that are able to convert phosphatidylinositol (PtdIns), PtdIns(4)P and PtdIns(4,5)P2 into PtdIns(3)P, PtdIns(3,4)P2 and PtdIns(3,4,5)P3 respectively, by phosphorylating the D-3 position of the inositol head groups of pho ...

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Purification of Chemokines from Natural Sources

Chemokines (e.g., IL-8) were originally identified as chemotactic proteins obtained from different natural sources. These were purified by the use of a bioassay followed by N-terminal sequencing and then molecular cloning giving the full sequence and making it possible to express the ch ...

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CFU-A Assay for Measurement of the Antiproliferative Effects of Chemokines on Murine Early Hemopoietic Progenitors

One of the more intriguing features of the hemopoietic system is the ability of a single phenotypically homogeneous stem cell to give rise—through differentiation and development—to all the phenotypically diverse mature immuno-hemopoietic cell types (1). The stem cell therefore s ...

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Glycosaminoglycan Binding Assays

The interaction of a select group of chemokines with glycosaminoglycans has been demonstrated previously, both by affinity coelectrophoresis (1) and by cell-surface binding assays and immobilized binding heparin assays (2). The significance of the interaction of chemokines wi ...

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Downmodulation and Recycling of Chemokine Receptors

Binding of chemokines to their receptors induces a variety of biological responses, ranging from early and rapid events such as receptor phosphorylation and calcium influx to prolonged responses such as cellular migration (1,2). Downmodulation of chemokine receptors from the cell s ...

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Analysis of Chemokine Receptor Endocytosis and Recycling

Endocytosis regulates the cell-surface expression of many seven transmembrane G-protein coupled receptors (7TM-GPCRs) and has been implicated in both desensitization following agonist-induced activation and resensitization (1,2). The internalization mechanism for ...

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26The Production of Chemokine Specific Monoclonal Antibodies: Chemokine RANTES

Monoclonal antibodies (MAbs) produced through the technique of somatic cell fusion are fundamental tools in the exploration of chemokine biology. This chapter details the procedures that were used to generate a panel of MAbs directed against the human chemokine RANTES (1,2). The general ...

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Chemokine Inhibition of HIV Infection

Human immunodeficiency virus (HIV) is a member of the retro virus family, classified under the lentivirus genus. Retroviruses are enveloped RNA viruses, which contain a core of capsid proteins, viral RNA, and enzymes. All infectious retroviral virions contain an enzyme, reverse transcr ...

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Monoclonal Antibodies to Chemokine Receptors

Monoclonal antibodies (MAbs) to chemokine receptors are providing remarkable insight into the roles these receptors play in basal leukocyte trafficking for immune response and surveillance as well as cell specific recruitment to sites of inflammation. This latter role in particu ...

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Targeted Expression of Chemokines In vivo

One of the main reasons for interest in chemokines is the ease with which their expression can be documented in physiological settings involving leukocyte trafficking or in diseases characterized by inflammatory cell infiltration. In the case of most ELR-containing CXC chemokines, t ...

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Chemokine Knockout Mice

As the chemokine family continues to grow in number, it is becoming increasingly important to determine the distinct function of each molecule. This is particularly important because of the apparent overlapping biological activity of some chemokines in vitro. One approach to determi ...

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