Flow Cytometry For Biotechnology

Flow cytometry is a sensitive and quantitative platform for the measurement of particle fluorescence. In flow cytometry, the particles in a sample flow in single file through a focused laser beam at rates of hundreds to thousands of particles per second. During the time each particle is in the laser beam, on the order of ten microseconds, one or more fluorescent dyes associated with that particle are excited. The fluorescence emitted from each particle is collected through a microscope objective, spectrally filtered, and detected with photomultiplier tubes. Flow cytometry is uniquely capable of the precise and quantitative molecular analysis of genomic sequence information, interactions between purified biomolecules and cellular function. Combined with automated sample handling for increased sample throughput, these features make flow cytometry a versatile platform with applications at many stages of drug discovery. Traditionally, the particles studied are cells, especially blood cells; flow cytometry is used extensively in immunology. This volume shows how flow cytometry is integrated into modern biotechnology, dealing with issues of throughput, content, sensitivity, and high throughput informatics with applications in genomics, proteomics and protein-protein interactions, drug discovery, vaccine development, plant and reproductive biology, pharmacology and toxicology, cell-cell interactions and protein engineering.

Flow cytometry has evolved since the 1940s into a multidisciplinary field incorporating aspects of laser technology, fluid dynamics, electronics, optics, computer science, physics, chemistry, biology, and mathematics. Innovations in instrumentation, development of small lasers, discovery of new fluorochromes/fluorescent proteins, and implementation of novel methodologies have all contributed to the recent rapid expansion of flow cytometry applications. In this thoroughly revised and updated second edition of Flow Cytometry Protocols, time-proven as well as cutting-edge methods are clearly and comprehensively presented by leading experimentalists. In addition to being a valuable reference manual for experienced flow cytometrists, the editors expect this authoritative up-to-date collection to prove useful to investigators in all areas of the biological and biomedical sciences who are new to the subject. The introductory chapter provides an eloquent synopsis of the principles and diverse uses of flow cytometry, beginning with a historical perspective and ending with a view to the future. Chapters 2–22 contain step-by-step protocols of highly practical and state-of-the-art techniques. Detailed instructions and helpful tips on experimental design, as well as selection of reagents and data analysis tools, will allow researchers to readily carry out flow cytometric investigations ranging from traditional phenotypic characterizations to emerging genomics and proteomics applications. Complementing these instructive protocols is a chapter that provides a preview of the next generation of solid-state lasers, and one that describes a rapid means to validate containment of infectious aerosols generated during high-speed sorting (Chapters 23–24).

"Flow Cytometry - Recent Perspectives" is a compendium of comprehensive reviews and original scientific papers. The contents illustrate the constantly evolving application of flow cytometry to a multitude of scientific fields and technologies as well as its broad use as demonstrated by the international composition of the contributing author group. The book focuses on the utilization of the technology in basic sciences and covers such diverse areas as marine and plant biology, microbiology, immunology, and biotechnology. It is hoped that it will give novices a valuable introduction to the field, but will also provide experienced flow cytometrists with novel insights and a better understanding of the subject.

Described here are the practical applications of flow cytometry in specific biological systems, ranging from cell biology to chromosome analysis and sorting. Three major areas of interest in cell and molecular biology are addressed: - Cell Activation and Biological Response. - Membrane-Ligand Interactions and Cell Identity. - Nuclear Components: Form and Function. Data management, expert systems and cell sorting techniques concerning all aspects of flow cytometry are also presented.

With rapid improvements in instrumentation, lasers, fluorophores, and data analysis software, flow cytometry is riding the crest of unprecedented, innovative advances. This thoroughly revised and up-to-date third edition of Flow Cytometry Protocols highlights the expanding contribution of flow cytometry to basic biological research and diagnostic medicine. Written by leading experts in the field, the book presents cutting-edge topics such as polychromatic, quantitative, and high throughput flow cytometry, novel multiparametric data analysis which breaks the dimensionality barrier, standard practice and safety measures for aerosol-generating cell sorting, conventional and imaging flow cytometry as well as minimalist imaging cytometry. As a volume in the highly successful Methods in Molecular Biology series, chapters contain brief introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and extensive notes on troubleshooting and avoiding known pitfalls. Authoritative and comprehensive, Flow Cytometry Protocols, Third Edition presents established as well as new flow cytometric methodologies in order to introduce beginning users to basic applications while opening new avenues of innovation for seasoned users.

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.

A much-needed primer on the use of laser flow cytometry for stem cell analysis Laser flow cytometry is a powerful tool for rapid analysis of cells for marker expression, cell cycle position, proliferation, and apoptosis. However, no resources specifically address the use of this methodology for the study of stem cells; this is especially important as stem cell analysis involves specialized methods and staining procedures based on specific characteristics such as marker expression, cell size, drug transport, and efflux of the stem cells. Now, this book reviews these procedures, discusses the science behind them, and provides real-world examples to illustrate the usefulness of the methods. It brings together world-class experts in pathology, biophysics, immunology, and stem cell research, who draw upon their extensive experience with the methods and show examples of good data to help guide researchers in the right direction. Chapter coverage includes: Stem cell analysis and sorting using side population Flow cytometry in the study of proliferation and apoptosis Stem cell biology and application Identification and isolation of very small embryonic-like stem cells from murine and human specimens Hematopoietic stem cells—issues in enumeration Human embryonic stem cells: long-term culture and cardiovascular differentiation Limbal stem cells and corneal regeneration Flow cytometric sorting of spermatogonial stem cells Breast cancer stem cells Stem cell marker expression in cells from body cavity fluids This book is an essential resource for all graduate students, practitioners in developing countries, libraries and book repositories of universities and research institutions, and individual researchers. It is also of interest to laboratories engaged in stem cell research and use of stem cells for tissue regeneration, and to any organization dealing in stem cell and tissue regeneration research.

From the reviews of the 3rd Edition... "The standard reference for anyone interested in understandingflow cytometry technology." American Journal of Clinical Oncology "...one of the most valuable of its genre and...addressed to awide audience?written in such an attractive way, being bothinformative and stimulating." Trends in Cell Biology This reference explains the science and discusses the vastbiomedical applications of quantitative analytical cytology usinglaser-activated detection and cell sorting. Now in its fourthedition, this text has been expanded to provide full coverage ofthe broad spectrum of applications in molecular biology andbiotechnology today. New to this edition are chapters on automatedanalysis of array technologies, compensation, high-speed sorting,reporter molecules, and multiplex and apoptosis assays, along withfully updated and revised references and a list of suppliers.