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Quantification of Adipose Tissue Leukocytosis in Obesity

The infiltration of immune cell subsets in adipose tissue termed “adipose tissue leukocytosis” is a critical event in the development of chronic inflammation and obesity-associated comorbidities. Given that a significant proportion of cells in adipose tissue of obese patients are ...

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Analysis of Microbiota Alterations in Inflammasome-Deficient Mice

Inflammasomes have emerged as central regulators of intestinal infection, immunity, and inflammation. Inflammasome activity mediates intestinal epithelial integrity, antimicrobial responses, and initiates inflammation through generation of the cytokines inte ...

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Blue Native Polyacrylamide Gel Electrophoresis to Monitor Inflammasome Assembly and Composition

Inflammasomes are large cytosolic multi-protein complexes that form in response to infection and other stimuli, and play an important role in the innate immune response. Traditional methods for assaying inflammasome activation have relied on detection of autoproteolysis of the c ...

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Assessing ATP Binding and Hydrolysis by NLR Proteins

Nucleotide-binding and leucine rich repeat domain-containing proteins (NLRs) are central to the formation of many inflammasome complexes. Several inflammasome forming NLR proteins are known to be ATPases, but the nucleotide binding specificity of many remains to be characteri ...

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Reconstituting the NLRP1 Inflammasome In Vitro

Nucleotide-binding domain leucine-rich-repeat containing receptors; NOD-Like Receptors (NLRs) were originally described as microbial sensors involved in host defense against pathogens that comprise an important component of the innate immune system. Recently, their ce ...

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Inflammasome Activation and Inhibition in Primary Murine Bone Marrow-Derived Cells, and Assays for IL-1, IL-1, and Caspase-1

Through its ability to control the proteolytic maturation and secretion of interleukin-1 family cytokines, the inflammasome occupies a central role in the activation of inflammation and also influences the shaping of adaptive immunity. Since it affects a multitude of different imm ...

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Human Leukocyte Antigen (HLA) Typing by DNA Sequencing

DNA sequencing is a powerful technique for identifying allelic variation within the human leukocyte antigen (HLA) genes. Sequencing is usually focused on the most polymorphic exons of the class I (HLA-A, -B, -C) and class II (HLA-DR, -DQ, and -DP) genes. These exons encode the antigen recognition s ...

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HLA Typing by Sequence-Specific Primers

HLA typing by sequence-specific primers (PCR-SSP) is a commonly used technique in HLA typing in which multiple pairs of cis-located allele-specific primers are used to determine the alleles present in a given DNA sample. Although the technique is around two decades old, it still offers a relati ...

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RNA Purification and Expression Analysis Using Microarrays and RNA Deep Sequencing

Transcriptome analysis or global gene expression profiling is a powerful tool for discovery as well as �understanding biological mechanisms in health and disease. We present in this chapter a description of methods used to isolate mRNA from cells and tissues that has been optimized for pre ...

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Discovery and Customized Validation of Antibody Targets by Protein Arrays and Indirect ELISA

Because of our access to human genome data and ever improving genome sequencing and proteome analysis methods we are much better in terms of our understanding of biological processes. In addition to genomics, proteomics, and other “omics” methods, availability of more sophisticated mol ...

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Tolerogenic Dendritic Cells and Induction of T Suppressor Cells in Transplant Recipients

Tolerogenic antigen presenting cells (APC), primarily dendritic cells (DC), are essential to the induction and maintenance of immunologic tolerance in clinical transplantation. They induce the differentiation of CD8+ T suppressor (Ts) and CD4+ T regulatory (Treg) or anergic cells, w ...

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Detection of Intracellular Cytokines

The intracellular cytokine method allows for a multiparametric readout by flow cytometry with precise phenotyping of the responding T cells. The intracellular cytokine staining of cells that have been fixed and permeabilized following a short-term activation and staining with an ...

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Cylex ImmuKnow Cell Function Assay

The use of immunosuppression in solid organ transplantation is associated with increased morbidity and mortality. Monitoring trough immunosuppression levels alone in transplant recipients is insufficient to gauge the suppression of immune responses. The ImmuKnow Immune C ...

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Detection of Antibodies to Self-Antigens (K-alpha 1 Tubulin, Collagen I, II, IV, and V, Myosin, and Vimentin) by Enzyme-Linked Immunosorbent Assay (EL

The enzyme-linked immunosorbent assay (ELISA) is a widely used technique for detecting antibodies (Abs) and is employed in clinical laboratories to identify Abs against various self-antigens–autoAb development and quantitation. This method relies on specific antigen–Ab int ...

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The Detection of Antibodies to the Angiotensin II-Type 1 Receptor in Transplantation

The detection of auto-antibodies detection is a major aspect of patient’s immunomonitoring. Antibodies directed against the heart and kidney Angiotensin II type 1 receptor (AT1R) play a major role in antibody mediated rejection after heart and kidney transplantation and in oblitera ...

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A Flow Cytometric Crossmatch Test Using Endothelial Precursor Cells Isolated from Peripheral Blood

Flow cytometric crossmatch tests provide a donor-specific, cell based method for the detection of alloreactive antibodies in the sera of transplant patients. Conventional crossmatch tests used in solid organ transplantation utilize lymphocytes as target cells to detect the pre ...

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Tetramer Staining for the Detection of HLA-Specific B cells

HLA-specific B cells can be identified, quantified, and isolated after staining with HLA tetramers. Quantification of these B cells can in turn identify individuals who are sensitized to HLA antigens and the isolation of these cells facilitates a variety of experimental investigation ...

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C1q Assay for the Detection of Complement Fixing Antibody to HLA Antigens

Solid phase Luminex� and flow cytometric single antigen bead assays offer exquisite sensitivity and specificity for HLA antibody detection. Unlike the historical complement-dependent cytotoxicity (CDC) method, these assays do not distinguish complement fixing from non-co ...

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Solid Phase Assay Measuring C4d Deposition to Determine Complement Fixation by HLA-Specific Antibodies

The definition of HLA-specific antibodies in solid organ transplant patients is a necessary tool for recipient selection prior to transplantation and monitoring for rejection post transplant. Solid phase assays can detect both complement fixing and non-complement fixing HLA-s ...

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Lymphocyte Crossmatching by Flow Cytometry

Flow cytometry is an invaluable tool for studying lymphocyte biology. In transplantation, flow cytometry represents the most sensitive method for demonstrating the binding of HLA antibodies to cellular targets. Referred to as the flow cytometric crossmatch (FCXM), the implement ...

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