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Detection of Dengue Virus

Global incidence of dengue has increased considerably over the past decade. Dengue fever (DF) is a self-limiting disease; however, dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS) are fatal. Since there is no therapy and vaccine against dengue, timely diagnosis is therefore n ...

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Detection of Human Caliciviruses in Fecal Samples by RT-PCR

Human caliciviruses, noroviruses in particular, are a common cause of gastroenteritis in persons of all age groups. Although both antigen detection and serologic methods for diagnosis of infection with these viruses have been described, the best and most common methods used for diagnos ...

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Innate Immune Receptors for Nucleic Acids

The innate immune system has evolved to detect microbes and sterile tissue damage with the help of a series of signaling receptors. One key strategy is to detect infectious microbes or host cell damage by recognizing nucleic acids that are modified or appear in compartment normally devoid of nuc ...

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Ratiometric Analysis of Subcellular Recruitment of Fc Receptors During Phagocytosis

Numerous immune receptors have the ability to mediate phagocytosis of large particles by triggering dynamic local rearrangement of the cytoskeleton and cell membrane. Different receptors can be differentially recruited to sites of particle binding, which in turn can have importa ...

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Determining FcRI Diffusional Dynamics via Single Quantum Dot Tracking

Single-particle tracking (SPT) using fluorescent quantum dots (QDs) provides high-resolution spatial–temporal information on receptor dynamics that cannot be obtained through traditional biochemical techniques. In particular, the high brightness and photostabil ...

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Cell-Based Reporter Assay to Analyze Activation of Nod1 and Nod2

Nod1 and Nod2 are pattern recognition receptors of the mammalian innate immune system. They respond to bacterial peptidoglycan fragments and are implicated in host defense against a variety of �different bacterial pathogens. Recent studies furthermore support additional func ...

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Analysis of ReceptorLigand Interactions by Surface Plasmon Resonance

Many immunological responses are often regulated by cell surface receptors in cell–cell recognition events. Such immune receptors on the cell surface typically exhibit low-affinity and fast-kinetic ligand interactions (e.g., K d in the μM range, k off  =  10−2 to 20 s−1). Real-time surface plasm ...

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NKT Cell Responses to Glycolipid Activation

NKT cells are a distinct lineage of T lymphocytes that are usually identified by the co-expression of the semi-invariant CD1d-restricted αβ TCR and the NK1.1 allelic marker of NK lineage receptors in the C57BL/6 mice and related strains. NKT cells can be subdivided based on CD4/CD8 expression and on ...

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In Vitro Effects of Adjuvants on B Cells

Stimulation of B cells not only through the B cell antigen receptor (BCR) but also through Toll-like receptors (TLRs) can drive activation, proliferation, and differentiation of B cells to result in antigen-specific antibody secretion. In addition, B cells are co-stimulated by specific a ...

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Probing the Plasma Membrane Structure of Immune Cells Through the Analysis of Membrane Sheets by Electron Microscopy

This chapter describes a method to generate plasma membrane sheets that are large enough to visualize the membrane architecture and perform quantitative analyses of protein distributions. This procedure places the sheets on electron microscopy grids, parallel to the imaging plane ...

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Measuring Immune Receptor Mobility by Fluorescence Recovery After Photobleaching

The coordinated effort of cells in the immune system relies heavily on surface receptor interactions. Immune receptor mobility provides vital information on the function and responses of immune cells, and these measurements shed light on their interactions with other membrane, cyt ...

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Assessment of the Recycling of the Membrane-Bound Chemokine, CX3CL1

Fractalkine (CX3CL1) is a membrane-anchored chemokine whose N-terminus contains a unique CX3C motif that is cleaved and released. The membrane-bound form functions as an adhesion molecule and the secreted form as a chemotactic factor. Like other chemokines, CX3CL1 is regulated at the le ...

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Construction, Expression, and Purification of Chimeric Protein Reagents Based on Immunoglobulin Fc Regions

Recombinant fusion proteins incorporating experimental protein domains fused to immunoglobulin Fc regions have become widely utilized in studies of protein–ligand interactions. The advantages of these systems include an inherent increase in avidity provided by the multim ...

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Identification of Scavenger Receptor Ligands

Scavenger receptors (SRs) are structurally diverse but functionally related innate immune receptors involved in defence and clearance mechanisms. Their broad specificity relies on evolutionarily conserved pattern recognition domains which interact with a variety of mic ...

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Yeast Surface Display of Lamprey Variable Lymphocyte Receptors

The variable lymphocyte receptors (VLRs) of lamprey and hagfish comprise leucine-rich repeat modules, instead of the immunoglobulin-like domain building blocks of antibodies and T-cell receptors in jawed vertebrates. Both types of vertebrate-rearranging antigen recepto ...

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Screening for Ligands of C-Type Lectin-Like Receptors

In order to execute their immune functions, leukocytes interact with a broad range of cell types through cell surface receptors, such as those of the immunoglobulin and C-type lectin families, or indirectly through soluble factors. The characterization of activating and inhibitory co ...

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Characterizing Immune Receptors from New Genome Sequences

Genome sequences are quickly being generated from a variety of organisms and provide researchers with an abundance of previously inaccessible information and an important source of insight into immune mechanisms. There are a variety of methods to accurately characterize genes from ...

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Characterizing Somatic Hypermutation and Gene Conversion in the Chicken DT40 Cell System

The secondary immunoglobulin gene diversification processes, somatic hypermutation (SHM), immunoglobulin gene conversion (GCV), and class switch recombination, are important for efficient humoral immune responses. They require the action of activation-induced cyti ...

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Transfection-Based Genomic Readout for Identifying Rare Transcriptional Splice Variants

Understanding the transcriptome, defined as the complete transcriptional component of the genome, is far more complex than originally considered. Even with the near fully resolved human and mouse genomes, for which extensive databases of transcribed sequence data (e.g., expressed ...

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Use of Colloidal Silica-Beads for the Isolation of Cell-Surface Proteins for Mass Spectrometry-Based Proteomics

Chaney and Jacobson first introduced the colloidal silica-bead protocol for the coating of cellular plasma membranes in the early 1980s. Since then, this method has been successfully incorporated into a wide range of in vitro and in vivo applications for the isolation of cell-surface prote ...

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