Preface (1998)

Although mastery of the techniques presented herein will allow the reader to design and complete experiments using immunological methods, this manual is not intended to be a substitute for an advanced program in immunology, nor for any of a number of comprehensive textbooks in the field (see recommended texts at the end of this preface). In addition to being thoroughly knowledgeable of the terms and concepts of immunology, we strongly recommend that readers gain first-hand experience in basic techniques and safety procedures by working in an immunology laboratory. An inevitable hazard of writing a methods manual is that protocols become obsolete or that new techniques are developed—usually just as the pages are going to press. This problem is particularly severe in a fast-moving field such as immunology. To safeguard our manual from inexorable obsolescence, and as a means of correcting errors in a timely manner, we provide a quarterly updating service. The looseleaf format will facilitate the replacement of pages that contain errors or have become obsolete and will accommodate the addition of new protocols to the appropriate section of the manual. The publisher can provide further details about subscribing to the quarterly supplements. We invite you to visit the Current Protocols home page for subscription information regarding this and other CP series (http://www.wiley.com/cp/cpi). This publication is available in both looseleaf and CD-ROM format. The material covered in the two versions is identical. For looseleaf purchasers, three binders are provided to accommodate the growth of Current Protocols due to quarterly supplementation. The looseleaf format allows easy insertion of new pages, units, and chapters that are added. The index and table of contents are updated with each supplement. CD-ROM purchasers receive a completely new disc containing the entire manual every quarter. The CD-ROM User's Guide describes in detail the many features designed for accessing information presented in the CD. Topics of interest can be located primarily by using either the search index (by typing in the desired word or string of words) or by scanning through the expandable table of contents and selecting the desired units. In addition, hyperlinks jump between related units when cross-references are selected and from solutions and reagents listed in the materials list to their corresponding recipes. Finally, hotlinks take you directly to appropriate web sites. Subjects are organized by chapters and sections, and protocols are contained in units. Units include listings of materials, the protocol steps, recipes for unique reagents and solutions, and commentaries on the “hows” and “whys” of the protocol. Page numbering reflects this modular arrangement; for example, page 7.5.2 refers to Chapter 7 (immunologic studies in humans), UNIT 5 (isolation of human B cell populations), page 2 of that particular unit. In some cases the numerical sequence of units within a given chapter may not reflect a logical order, because of the manner in which these chapters developed over time. Many reagents and procedures are employed repeatedly throughout the manual. Rather than duplicate this information, cross-references among units are used extensively. Early chapters (and appendices) describe these commonly used techniques (care and handling of laboratory animals, and basic procedures for production of antibodies and isolation of specific cell types), while later chapters describe their application (assays for B and T cell function, measurement of lymphokines and their receptors, flow cytometry, protein analysis, and peptide synthesis). Thus, whenever it is necessary to use an ELISA in a protocol, the appropriate unit in Chapter 2—describing the procedure for an ELISA—is cross-referenced (e.g., UNIT Unavailable for several different ELISAs). Similarly, throughout the book readers are referred to UNIT Unavailable for removal of lymphoid organs, to UNIT Unavailable for production of monoclonal antibodies, to UNIT Unavailable for isolation of mouse lymphocytes, and so on. By turning to these units, the reader will find instructions for the techniques, recipes for relevant reagents, and commentary. As a result, protocols in the later chapters of the book—which can be lengthy and complex—are not overburdened with steps describing auxiliary procedures required to prepare, purify, and analyze the sample or molecule of interest. Because this publication is first and foremost a compilation of immunology techniques, we have not provided extensive instructive material. We have, however, included explanatory information where required to help readers gain an intuitive grasp of the procedures. First, the chapter, section, and unit introductions describe how the protocols that follow connect to each other. Second, annotations to the actual protocol steps describe what is happening. Third, the commentary that closes each unit gives background information and reviews the relevant literature so that readers are aware of the historical and theoretical development of a technique, as well as alternative approaches; in addition, critical information and troubleshooting guidelines are presented as well as anticipated results and time considerations. Fourth, key references and Internet sites are sometimes included to inform users of particularly useful background reading or original descriptions of the technique. Finally, in some cases special “overview” units have been included early in the chapter. 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 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 separately from the basic protocol steps. Reagents required for a protocol are listed 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., blocking buffer, lysis buffer, etc.) while the recipes differ; thus, make certain that reagents are prepared from the proper recipes. On the other hand, recipes for commonly used buffers and media are provided once in an appendix or in early units in the manual. These universal recipes are cross-referenced parenthetically in the materials lists rather than repeated with every usage. NOTE: Deionized, distilled water should be used in all protocols in this manual, and in the preparation of all reagents and solutions. Protocols requiring sterile technique are so noted. 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 immunologic experimenter in obtaining the tools of the trade. Experienced investigators are therefore encouraged to experiment with substituting their own favorite brands. The Appendix section is a rich resource containing sections with protocols on common immunologic techniques, recipes for commonly used reagents, media, and stock solutions, abbreviations and useful data, and a comprehensive table of CD antigens in the human, mouse, and rat. Finally, addresses, phone numbers, and facsimile numbers of all suppliers mentioned in this manual are provided in APPENDIX Unavailable. Also recommended is an updated sourcebook of immunologic reagents and suppliers, Linscott's Directory of Immunological and Biological Reagents (Santa Rosa, Calif.). Anyone carrying out these protocols will encounter the following hazardous or potentially hazardous materials: (1) radioactive substances, (2) toxic chemicals and carcinogenic or teratogenic reagents, (3) pathogenic and infectious biological agents, and (4) recombinant DNA. Most governments regulate the use of these materials; it is essential that they be used in strict accordance with local and national regulations. Cautionary notes are included in many instances throughout the manual (in particular, see the introductions to Chapter 7 for consideration of the hazards of working with human material and Chapter 12 for hazards associated with studies of HIV), but we emphasize that users must proceed with the prudence and precaution associated with good laboratory practice. Radioactive substances must of course be used only under the supervision of licensed users, following guidelines of the Nuclear Regulatory Commission (NRC). 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. Consequently, a self-mailing, reader-response survey is included at the back of the manual (and is included with each supplement); we encourage readers to send in their comments. Richard Coico CPI Series Editor Department of Microbiology and Immunology City University of New York Medical School Convent Avenue at 138th Street New York, NY 10031 Alternatively, letters of intent may be submitted by e-mail to [email protected]. Additional instructions to authors interested in submitting letters of intent can be found on our web site (http://wiley.com/cp/cpi). Assembling this manual required extensive knowledge and effort, primarily by Sarah Greene, who initially conceived this project and helped to shape it with skill and unbounded patience. Continuing to keep this manual up to date would be impossible without massive assistance from the Current Protocols staff at John Wiley & Sons. Among those who help us, we are extremely grateful to Kathy Wisch, Janet Blair, Alice Ro, Rebecca Barr, Allen Ranz, Elizabeth Harkins, Benjamin Gutman, Michael Gates, and Liana Scalettar. In addition, we thank our illustrators, Gae Xavier Studios, Jo Moore, and Jennifer Steinmetz, for visually conveying details that defy verbal communication. Finally, we would like to extend our gratitude and thanks to Richard Coico, the Series Editor, who plays a critical role in helping us implement the editorial content of this series and in managing the day-to-day activity associated with its maintenance. Several individuals offered helpful critical advice at various stages in the development of this manual. Jeffrey Bluestone read, pointedly commented on, and provided additions to the entire text. Individual chapters were thoughtfully critiqued by Kenneth A. Ault, John H. Freed, Rosemarie Hunziker, Marc K. Jenkins, Ralph T. Kubo, John T. Kung, Peter E. Lipsky, Jan Lukszo, David C. Parker, Robert R. Rich, Andrea J. Sant, Clifford E. Snyder, Susan L. Swain, Lucio Tentori, William F. Wade, and Henry Wortis. We are particularly grateful to our colleagues who have contributed the material comprising the manual, not only for their protocols and laboratory experience, but for their patience with repeated editorial queries and for their supportive enthusiasm for the entire project. As well, we thank the members of our own laboratories and our immunologist friends throughout the world who have given unsolicited encouragement and help.