In this first book dedicated entirely to the ELISPOT, a critical enzyme-linked immunospot assay used widely in biomedical research, recognized experts with first-hand experience detail how to design, perform, and analyze these assays. The readily reproducible techniques they provide cover a wide variety of topics, including the use of membrane-backed plates, the standardization and validation procedures, the removal of cells from ELISPOT plates, cell separation techniques, and the quantification of ELISPOT data. There are also numerous ELISPOT applications involving animal models, human cells, measles, multiple sclerosis, immune responses, multicytokine detection systems, and immunocytochemistry. Highlights include dual-color and multiplex ELISPOT assays, use of the ELISPOT assay on feline lymphocytes, standardization of the ELISPOT procedure, and combining the ELISPOT assay with immunohistochemistry.

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 multiple cytokines 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.

Since the publication of the first edition of the Handbook of Human Immunology in 1997, major scientific achievements have directly contributed to an increased understanding of the complexities of the human immune system in health and disease. Whether as a result of the sequencing of the entire human genome, or of technological advancements, severa

This first edition volume demystifies the complex topic of flow cytometry by providing detailed explanations and nearly 120 figures to help novice flow cytometry users learn and understand the bedrock principles necessary to perform basic flow cytometry experiments correctly. The book divides the topic of flow cytometry into easy to understand sections and covers topics such as the physics behind flow cytometry, flow cytometry lingo, designing flow cytometry experiments and choosing appropriate fluorochromes, compensation, sample preparation and controls and ways to assess cellular function using a variety of flow cytometry assays. Written as a series of chapters whose concepts sequentially build off one another, using the list of materials contained within each section along with the readily reproducible laboratory protocols and tips on troubleshooting that are included, readers should be able to reproduce the data figures presented throughout the book on their way to mastering sound basic flow cytometry techniques. Easy to understand and comprehensive, Flow Cytometry Basics for the Non-Expert will be a valuable resource to novice flow cytometry users as well as experts in other biomedical research fields who need to familiarize themselves with a basic understanding of how to perform flow cytometry and interpret flow cytometry data. This book is written for both scientists and non-scientists in academia, government, biotechnology, and medicine.

Edited by renowned protein scientist and bestselling author Roger L. Lundblad, with the assistance of Fiona M. Macdonald of CRC Press, this fourth edition of the Handbook of Biochemistry and Molecular Biology represents a dramatic revision — the first in two decades — of one of biochemistry's most referenced works. This edition gathers a wealth of information not easily obtained, including information not found on the web. Offering a molecular perspective not available 20 years ago, it provides physical and chemical data on proteins, nucleic acids, lipids, and carbohydrates. Presented in an organized, concise, and simple-to-use format, this popular reference allows quick access to the most frequently used data. Covering a wide range of topics, from classical biochemistry to proteomics and genomics, it also details the properties of commonly used biochemicals, laboratory solvents, and reagents. Just a small sampling of the wealth of information found inside the handbook: Buffers and buffer solutions Heat capacities and combustion levels Reagents for the chemical modification of proteins Comprehensive classification system for lipids Biological characteristics of vitamins A huge variety of UV data Recommendations for nomenclature and tables in biochemical thermodynamics Guidelines for NMR measurements for determination of high and low pKa values Viscosity and density tables Chemical and physical properties of various commercial plastics Generic source-based nomenclature for polymers Therapeutic enzymes About the Editors: Roger L. Lundblad, Ph.D. Roger L. Lundblad is a native of San Francisco, California. He received his undergraduate education at Pacific Lutheran University and his PhD degree in biochemistry at the University of Washington. After postdoctoral work in the laboratories of Stanford Moore and William Stein at the Rockefeller University, he joined the faculty of the University of North Carolina at Chapel Hill. He joined the Hyland Division of Baxter Healthcare in 1990. Currently Dr. Lundblad is an independent consultant and writer in biotechnology in Chapel Hill, North Carolina. He is an adjunct Professor of Pathology at the University of North Carolina at Chapel Hill and Editor-in-Chief of the Internet Journal of Genomics and Proteomics. Fiona M. Macdonald, Ph.D., F.R.S.C. Fiona M. Macdonald received her BSc in chemistry from Durham University, UK. She obtained her PhD in inorganic biochemistry at Birkbeck College, University of London, studying under Peter Sadler. Having spent most of her career in scientific publishing, she is now at Taylor and Francis and is involved in developing chemical information products.

The recent explosion of information in innate immune pathways for recognition, effect or responses, and genetic regulation has given impetus to investigations into analogous pathways in the human immune response, which in turn has produced attendant insights into both normal physiology and immunopathology. This volume presents a compendium of methods and protocols for the investigation of human innate immunity with application to the study of normal immune function, immunosenescence, autoimmunity and infectious diseases. Among the topics covered are quantitative flow cytometry for Toll-like receptor expression and function; multidimensional single cell mass cytometry (CyTOF) in complex immune interactions and tumor immunity; imaging techniques such as Imagestream high resolution microscopy coupled to flow cytometry, immune cell infiltration of organotypic, biomimetic organs; high-throughput single cell secretion profiling; multiplexed transcriptomic profiling; microsatellite and microRNA methodologies, RNA interference; and the latest bioinformatics and biostatistical methodologies, including in-depth statistical modeling, genetic mapping, and systems approaches.