Preface

Abstract

Flow cytometry is not a new field: it has existed for about 30 years and would be considered a mature technology by most scientists. However, it is continually rediscovered by young researchers who recognize its potential to answer many diverse questions. So wide-ranging is the field that its practitioners include engineers, physicians, microbiologists, marine biologists, veterinarians, and computer scientists, among others. In all of these cases, the technology requires precise application in order to obtain verifiable results, a task that is well within its capabilities. For example, it is currently possible to determine the number of antibody molecules actually bound to a cell rather than just the percentage of “positive” cells. Fluorescent indicators of calcium ions can identify changes in calcium fluxes on specific subsets of lymphocytes. Simultaneous multivariate analysis is now standard practice in clinical immunophenotyping. Cells expressing a specific phenotype can be identified, quantified, and isolated using reporters such as green fluorescent protein and the sorting capability of flow cytometry. Microdissected or flow-sorted abnormal chromosomes may be characterized by “reverse painting” following universal amplification strategies. Cytometrists continue to find and develop new applications for both flow and image cytometry. For example, confocal microscopes and digital imaging systems, like flow cytometers, allow simultaneous detection of multiple fluorochromes. Understanding the links between these two technologies makes it clear that they are complementary in many ways. For this reason, an attempt has been made in this manual to combine flow and image commentary and protocols to some extent. Technologies common to both, such as optical filters, light sources, and molecular probes, are given particular attention. With regard to protocols, the initial approach has been to emphasize flow cytometry, with a commitment to increase the image cytometry content in subsequent updates. This manual is designed to provide a definitive source of comprehensive methods, entirely updatable for a field that continues to grow and spread its influence. Because the field is so diverse, a wide range of techniques must often be considered and tested empirically for a particular application, and each researcher's arsenal must include both recent innovations and techniques that have been used for many years. The manual includes a mixture of descriptive information covering such topics as equipment, strategies, and data analysis; specific detailed protocols; and approaches for adapting methods to particular projects. It is designed to meet the needs of scientists with little prior experience of flow cytometry, including graduate students and scientists trained in other biological disciplines. At the same time, the broad range of techniques presented here should ensure that even seasoned experts will find new and useful approaches and will benefit from the manual's convenient compilation of standard information and methods that typically must be culled from many sources. Quarterly updates will cover emerging capabilities, keep up with the full range of current techniques, and provide new commentaries on expanding technologies. Subjects in this manual are organized by chapters, which are in turn broken down into units. Protocol units generally describe a method and include one or more protocols with listings of materials, step-by-step instructions with annotations, recipes for unique reagents and solutions, and commentaries on the “hows” and “whys” of the method to help readers gain an intuitive grasp of the procedures. Overview units contain theoretical discussions that lay the foundation for subsequent protocols. Commentary units present more general information in the form of explanatory text with no protocols: for example, Chapter 10 contains several units that present advanced material on data management and processing to which the reader may turn to gain a greater understanding of experimental results. Page numbering reflects the modular arrangement by unit; for example, page 9.3.5 refers to Chapter 9 (Studies of Cell Function), UNIT Unavailable (Measurements of Intracellular pH), page 5 of that particular unit. Many reagents and procedures are employed repeatedly throughout the manual. Instead of duplicating this information, cross-references among units are used extensively. Cross-referencing helps to ensure that lengthy and complex protocols are not overburdened with steps describing auxiliary procedures needed to prepare raw materials and analyze results. Certain units that describe commonly used techniques and recipes (e.g., handling, storage, and preparation of human blood, nucleic acid probes) are cross-referenced in other units that describe their application (e.g., differential staining of DNA and RNA). For some widely used techniques (such as counting cells), readers are referred to APPENDIX Unavailable. Relevant commentary is cross-referenced similarly; for example, whenever specialized information on data management and processing is relevant, the appropriate units in Chapter 10 are referred to. Chapters 1 and 2 introduce the technology of flow cytometry and image analysis respectively. The units in these chapters are commentaries designed to provide an understanding of the basis of the technology. Some subsequent chapters begin with special overview units that describe the technological state of the art vis-à-vis the topic at hand and provide a context for the procedures that follow. Chapter and unit introductions describe how the protocols that follow connect to one another, and annotations to the actual protocol steps describe what is happening as a procedure is carried out. Finally, the Commentary that closes each protocol unit describes background information regarding the historical and theoretical development of the method, as well as alternative approaches, critical parameters, troubleshooting guidelines, anticipated results, and time considerations. All units contain cited references and many indicate key references to inform users of particularly useful background reading, original descriptions, or applications of a technique. Many units in the manual contain groups of protocols, each presented with a series of steps. The basic protocol is presented first in each unit and is generally the recommended or most universally applicable approach. Alternate protocols are provided where different equipment or reagents can be employed to achieve similar ends, where the starting material requires a variation in approach, or where requirements for the end product differ from those in the basic protocol. Support protocols describe additional steps that are required to perform the basic or alternate protocols; these steps are separated from the core protocol because they might be applicable to other uses in the manual, or because they are performed in a time frame separate from the basic protocol steps. Reagents required for a protocol are itemized in the materials list before the procedure begins. Many are common stock solutions, others are commonly used buffers or media, while others are solutions unique to a particular protocol. Recipes for the latter solutions are provided in each unit, following the protocols (and before the commentary) under the heading Reagents and Solutions. It is important to note that the names of some of these special solutions might be similar from unit to unit (e.g., SDS sample buffer) while the recipes differ; thus, make certain that reagents are prepared from the proper recipes. On the other hand, recipes for commonly used stock solutions and buffers are provided once in APPENDIX Unavailable. These universal recipes are cross-referenced parenthetically in the materials lists rather than repeated with every usage. Throughout the manual, we have recommended commercial suppliers of chemicals, biological materials, and equipment. In some cases, the noted brand has been found to be of superior quality or it is the only suitable product available in the marketplace. In other cases, the experience of the author of that protocol is limited to that brand. In the latter situation, recommendations are offered as an aid to the novice experimenter in obtaining the tools of the trade. Experienced investigators are therefore encouraged to experiment with substituting their own favorite brands. Anyone carrying out these protocols may encounter toxic chemicals, carcinogenic or teratogenic reagents, and pathogenic and infectious biological agents. Particular care should be exercised in dealing with fluorescent probes, many of which are potential carcinogens, especially when sorting cells labeled by or suspended in fluorescent reagents. All applicable precautions must be taken for any work with materials of human origin. Although cautionary statements are included in the appropriate units, it must be emphasized that users must proceed with the prudence and precaution associated with good laboratory practice, and that all materials must be used, stored, and disposed of in strict accordance with federal, state, and local regulations. Most of the protocols included in this manual are used routinely in our own laboratories. These protocols work for us; to make them work for you we have annotated critical steps and included critical parameters and troubleshooting guides in the commentaries to most units. However, the successful evolution of this manual depends upon readers' observations and suggestions. We encourage readers to send their comments to currentprotocols@wiley.com. Another valuable resource is the companion website http://www.currentprotocols.com. This site features tools, calculators, apps, troubleshooting tips, webinars, and videos to assist in the experiments presented in this manual. The individual article pages provide valuable summary information and easy access to the content. While the editorial board enlists contributors for most of the topics presented in this manual, we invite individuals to submit letters of intent describing new topics that they would like to publish in CPCY. The editorial board will carefully consider suggested topics and invite authors to submit full manuscripts in some cases. Letters of intent may be submitted by email to currentprotocols@wiley.com. This manual is the product of dedicated efforts by many of our scientific colleagues who are acknowledged in each unit and by the hard work of the Current Protocols editorial staff at John Wiley and Sons. Skilled members of the Current Protocols staff who contributed to this effort include Janet Blair, Virginia Chanda, Michael Gates, Scott Holmes, and Kathy Wisch. The extensive copyediting required to produce an accurate protocols manual was ably handled by Jill Adams, Rebecca M. Barr, Elizabeth Harkins, Kathy Morgan, Constance Parks, Allen Ranz, Beverly Rothermel, and Kristine Templeman; electronic illustrations were prepared by Gae Xavier. Finally, we would like to thank Gretchen Lawler at the Purdue University editorial offices. Her attention to the hundreds of details, as well as her constant reminders to authors and editors, were crucial to the appearance of this manual. Provides broad coverage of flow cytometry methods with detailed explanations. The most comprehensive available reference on cytometry. Detailed coverage of the technology and fluorescent probes, covering a little of everything. A comprehensive survey of data analysis from first principles to practical applications. Broad coverage of clinical and closely related research in flow cytometry. Easy reading for the clinical practitioner, with good coverage of immunophenotyping and DNA applications. A very comprehensive volume that takes into account the perspectives of both practitioners and laboratory technologists. A practical manual for research applications. An excellent book to have in the lab, providing for confocal microscopy what Shapiro's book provides for flow cytometry. An indispensable reference book for anyone working in image analysis. Provides applications-oriented discussion of confocal microscopy.