T Cell Protocols (2^nd Ed., 2nd ed. 2009) Methods in Molecular Biology Series, Vol. 514

This book is a collection of protocols, to provide novel techniques for the study of the biology of T lymphocytes. The methods described in this book do not cover all of the techniques currently used to study T cell-mediated immune responses for the simple reason that T cell immunology is probably the immunological discipline which can be investigated with the widest variety of approaches. Thechoiceofchapterswasmadetakingintoaccounttwopoints:First,manyofthe techniquesthathavebeen usedfor sometimehave beenupgraded duringthe pastfew years given the greater availability of a variety of products (i.e. cytokines, chemokines, monoclonal antibodies), of refined technical devices (i.e. novel cell culture and cell analysis equipments), and the development of novel instrumentation (i.e. multipa- metric flow cytometers, confocal microscopes). Therefore, in several chapters ??old techniques??, which remain fundamental to T cell immunology, are described in their ??modern?? versions. Secondly, the technical advancement has generated the possibility to establish novel assays to investigate T cell physiology. This is reflected in the chapters which describe the protocols that allow use of these modern approaches. The preparation of this book has required participation of several scientists, all leading experts in their respective fields. Without their enthusiastic participation, this work would not have been possible. Therefore, I thank all authors for their contri- tionsandacceptallcriticismformissingparts,orinformationordetails,forwhichonly I am responsible. I hope these protocols will be useful for young investigators who approach for the first time the complex field of immunology and for those more experienced scientists who look for concise and efficaciousdescriptions of novel methods.

Analysis of Frequency and Phenotype of Antigen-Specific T Cells.- B Cell Helper Assays.- transkingdom RNA Interference (tkRNAi): A Novel Method to Induce Therapeutic Gene Silencing.- Flow Cytometry and Cell Activation.- Investigating T Cells by Polychromatic Flow Cytometry.- Generation of Human T Cell Clones.- Limiting Dilution Analysis of Antigen-Specific T Cells.- T Cell Epitope-Mapping by Cytokine Gene Expression Assay.- Cytokine Multiplex Immunoassay: Methodology and (Clinical) Applications.- Purification of the T Cell Antigen Receptor and Analysis by Blue-Native PAGE.- Non-Replicating Recombinant Vaccinia Virus Expressing CD80 to Enhance T-Cell Stimulation.

Provides an easily accessible reference volume for cell-based phenotypic screening, written by leading researchers in the field Covers a wide range of cell-based assay formats and systems Presents a modular approach to experimental design, covering model biological systems, screening modalities, detection technologies, and approaches to data analysis Comprehensive guide to developing, running, and analyzing cell-based assays