Cytokines are pleiotropic regulatory proteins involved in essentially all biological processes and associated with a wide variety of diseases, including inflammatory disorders as well as many types of cancer and leukemia. Knowledge about the quantitative and qualitative nature of cytokine production is critical in the understanding of normal and pathological processes. The cytokine detection in biological and clinical samples faces many challenges including their low abundance, the need to distinguish between active and latent cytokine forms, and the need to measure multiple cytokines in a single assay. This volume will provide a comprehensive collection of classic and cutting-edge methodologies that are currently used to analyze and quantify cytokines and their biological activities in complex biological and clinical samples. The chapters are divided into four main categories. The first group focuses on the immunodetection of released cytokines in tissue culture supernatants, plasma, serum and whole blood samples by immunoassays. These immunoassays measure the total concentrations of released cytokines regardless of their biological activity and include ELISA, flow cytometry, ELISPOT and the antibody-based proximity ligation. The second group will focus on the analysis of biologically active cytokines by bioassays using neutralizing antibodies, chemotaxis assay, cytokine-induced cell degranulation assay, cell proliferation and differentiation, cytokine-induced cytokine production and the radioreceptor cytokine assay. The third group focuses on the analysis of intracellular cytokines by flow cytometry, western blotting and fluorescence and confocal microscopy. In addition, this category includes protocols for quantitative analysis of cytokine gene expression by real time RT-PCR and analysis of the cytokine promoter occupancy by chromatin immunoprecipitation. The fourth group focuses on the recently developed multiplex arrays that can measure multiplecytokines in the same sample at the same time. This group includes quantification of multiple cytokines using cytometric bead arrays, ELISPOT assays, proteomics cytokine evaluation, multiplexed proximity ligation assays for high-throughput cytokine analysis and finally, cytokine gene expression analysis by gene arrays. The protocols will be written by experienced basic and clinical researchers with hands-on knowledge of the described protocols. By covering a broad variety of methods used in cytokine detection and analysis, this book will be of interest not only to biochemists, molecular biologists and immunologists but also to physician-scientists working in the field of cytokine research.

Includes cutting-edge methods and protocols Provides step-by-step detail essential for reproducible results Contains key notes and implementation advice from the experts Includes supplementary material: sn.pub/extras

Klappentext

Cytokine Bioassays: Methods and Protocols provides a comprehensive collection of classic and cutting-edge methodologies that are used to analyze and quantify cytokines and their biological activities in complex biological and clinical samples. Chapters are divided into three main categories, the first category details the immunodetection of released cytokines in tissue culture supernatants, plasma, serum, and whole blood samples by immunoassays. The second part focuses on the analysis of biologically active cytokines by bioassays using neutralizing antibodies, chemotaxis assay, cytokine-induced phagocytosis assay, proteasome activity assay, and analysis of cytokine-induced immunoglobulin class switching. Part three presents analysis of intracellular cytokines by flow cytometry, immunohistochemistry, immunofluorescence confocal microscopy, and western blotting. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and key tips on troubleshooting and avoiding known pitfalls.

Authoritative and practical, Cytokine Bioassays: Methods and Protocols is written by experienced, basic, and clinical researchers with hands-on knowledge of the described protocols. This book will be of interest not only to biochemists, molecular biologists and immunologists but also to physician-scientists working in the field of cytokine research.

Inhalt
The Challenge of Measuring Elusive Immune Markers by Enzyme Linked Immuno-Spot (ELISPOT) Technique.- Analysis of the Released Nuclear Cytokine HMGB1 in Human Serum.- ELISA Based Assay for IP-10 Detection from Filter Paper Samples.- Enhanced ELISA Based on Carboxymethylated Dextran Coatings.- A Novel Three-Dimensional Biosensor Based on Aluminum Oxide: Application for Early Stage Detection of Human Interleukin-10.- Quantification of Multiple Cytokines and Chemokines Using Cytometric Bead Arrays.- A Useful Guide for Analysis of Immune Markers by Fluorochrome (Luminex) Technique.- Control of Pro-inflammatory Cytokine Release from Human Monocytes with the Use of an Interleukin-10 Monoclonal Antibody.- Cytokine-Induced Neutrophil Chemotaxis Assay.- An In Vitro One-Dimensional Assay to Study Growth Factor-Regulated Tumor Cell-Macrophage Interaction.- Chemotactic Responses by Macrophages to a Directional Source of a Cytokine Delivered by a Micropipette.- Assessment of Phagocytic Activity of Cultured Macrophages Using Fluorescence Microscopy and Flow Cytometry.- Assessment of Cytokine-Modulated Proteasome Activity.- Evaluation of the Adverse Effect of Low Concentration of Cadmium on Interleukin-4 Induced Class Switch Recombination in Burkett's Lymphoma Raji Cell Line.- A Mix-and-Measure Assay for Determining the Activation Status of Endogenous Cdc42 in Cytokine-Stimulated Macrophage Cell Lysates.- Analysis of the Cell Surface Expression of Cytokine Receptors Using the Surface Protein Biotinylation Method.- Detection of CXCR2 Cytokine Receptor Surface Expression Using Immunofluorescence.- Detecting Tie2, an Endothelial Growth Factor Receptor, by Using Immunohistochemistry in Mouse Lungs.- Use of shRNA to Stably Suppress Chemokine Receptor Expression and Function in Human Cancer Cell Lines.- Intracellular Staining and Detection of Cytokines by Fluorescence-Activated Flow Cytometry.- Cytokine Detection by Flow Cytometry.- Analysis of IL-17 Production by FlowCytometry and ELISPOT Assays.- Interleukin-1 (IL-1) Immunohistochemistry Assay in Oral Squamous Cell Carcinoma.- Immunofluorescence Confocal Microscopy of Intracellular TNF in Human Neutrophils.- Evaluating Cytoplasmic and Nuclear Levels of Inflammatory Cytokines in Cancer Cells by Western Blotting.- Western Analysis of Intracellular Interleukin-8 in Human Mononuclear Leukocytes.- Quantitative Analysis of Bortezomib Induced IL-8 Gene Expression in Ovarian Cancer Cells.- Analysis of TGFb1 and IL-10 Transcriptional Regulation in CTCL Cells by Chromatin Immunoprecipitation.- Radiolabeled Semi-Quantitative RT-PCR Assay for the Analysis of Alternative Splicing of Interleukin Genes.