Preface

Abstract

The universe of cell biology is expanding. One way to sense this expansion is to attend a major meeting dedicated to the field, such as that of the American Society for Cell Biology. The poster sessions allow one to take a leisurely stroll through the exhibits and, even without stopping to look at any single poster, get a very good visceral feel for the scope of cell biology. For those who have taken such a stroll periodically over the past decade or two, the dynamic nature of cell biology and its explosive growth have been obvious—there is simply more and more about more and more. Once upon a time, poster sessions were dominated by images of cells fixed (literally) and captured in black and white by electron microscopists. While the electron microscope continues to contribute to our understanding of cell architecture, more recent poster sessions have witnessed the emergence of row upon row of television monitors where one can view computer-enhanced video images of living cells captured in the not-so-living colors of rhodamine red, fluorescein yellow, and the characteristic green that gives the now-famous fluorescent protein its name. Freeze-etching has been joined by freeze-frame. Furthermore, interspersed among the television sets and whirring VCRs are posters that document detailed molecular characterizations of a vast array of biochemical components that comprise the diverse cell types being investigated. Other posters record the latest genes to fall to the seemingly relentless onslaught of the cloners and sequencers, and still others offer functional insights obtained by knocking out these genes. Every point of the compass has its blot! Genetic studies in yeast, flies, and worms abound on the one hand, and on the other, a growing number of the presentations form bridges between basic research in cell biology and the practice of clinical medicine. Clearly, the scientists who today identify themselves as cell biologists are a diverse community, and great value resides in that diversity. Borders that once separated research disciplines have faded, and cell biologists have come to appreciate that no single approach in isolation will open the profound mysteries of the cell. New techniques and new technologies come alongside the tried-and-true as the tools of cell biology expand along with the field. It is this changing face of cell biology and its methodologies that represented the greatest challenge in pulling together Current Protocols in Cell Biology. A foundational question addressed by the editors of this work involved where to draw the boundaries around the field of cell biology. Our decision has been to refuse to draw such boundaries—they are artificial at best and counterproductive at worst. Instead, we will attempt in this effort to match the diversity of our field. We will include “classic” methods that remain valuable tools for the modern cell biologist and will also provide protocols that we believe are destined to become tomorrow's classics. There is no reason to suspect that the expanding universe of cell biology will cease expanding anytime soon. Indeed, part of the thrill of being a cell biologist is being constantly surprised by new innovations and discoveries. As a consequence, however, our community needs a reliable and user-friendly source of laboratory methods that is as expansive as the field itself. To begin to address this need, we have assembled a series of protocols that cover many aspects of cell biology. While this set of methods is incomplete, it can be considered a “starter toolbox” that includes many of the most versatile and essential instruments of our trade. This collection will expand as regular quarterly supplements are added to Current Protocols in Cell Biology. Through these supplements, the editors will endeavor to make the toolbox increasingly more useful over time. Because of the rapid pace of innovation and discovery in cell biology, we are expecting that our efforts will be met with some degree of very pleasant frustration as we strive to keep up with this fast-moving and exciting field. Subjects in this manual are organized by chapters, and protocols are contained in units. Protocol units, which constitute the bulk of the book, generally describe a method and include one or more protocols with listings of materials, steps and annotations, recipes for unique reagents and solutions, and commentaries on the “hows” and “whys” of the method. Other units present more general information in the form of explanatory text with no protocols. Overview units contain theoretical discussions that lay the foundation for subsequent protocols. Other discussion units present more general information. Page numbering reflects the modular arrangement by unit; for example, page 1.2.3 refers to Chapter 1 (Cell Culture), UNIT Unavailable (Media for Culture of Mammalian Cells), page 3 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 and recipes for common reagents are supplied in APPENDIX Unavailable. 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. Because this publication is first and foremost a compilation of laboratory techniques in cell biology, we have included explanatory information where required to help readers gain an intuitive grasp of the procedures. Some chapters begin with special overview units that describe the state of the art of the topic matter 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. One or more basic protocols are presented first in each unit and generally cover the recommended or most universally applicable approaches. 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., RIPA 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 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 the following hazardous or potentially hazardous materials: (1) radioactive substances, (2) toxic chemicals and carcinogenic or teratogenic reagents, and (3) pathogenic and infectious biological agents. Check the guidelines of your particular institution with regard to use and disposal of these hazardous materials. Although cautionary statements are included in the appropriate units, we emphasize that users must proceed with the prudence and precaution associated with good laboratory practice, and that all materials must be used in strict accordance with local and national regulations. Many protocols call for use of live animals (usually rats or mice) for experiments. Prior to conducting any laboratory procedures with live subjects, the experimental approach must be submitted in writing to the appropriate Institutional Animal Care and Use Committee (IACUC) or must conform to appropriate governmental regulations regarding the care and use of laboratory animals. Written approval from the IACUC (or equivalent) committee is absolutely required prior to undertaking any live-animal studies. Some specific animal care and handling guidelines are provided in the protocols where live subjects are used, but check with your IACUC or governmental guidelines to obtain more extensive information. Most of the protocols included in this manual are used routinely in the authors' laboratories. These protocols work for them; to make them work for you they 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 CPCB. 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 by the Current Protocols editorial staff at John Wiley and Sons. We are extremely grateful for the critical contributions by Kathy Morgan (Series Editor) who kept the editors and the contributors on track and played a key role in bringing the entire project to completion. Other skilled members of the Current Protocols staff who contributed to the project include Joseph White, Janet Blair, Kathy Wisch, Michael Gates, Demetra Kagdis, Alice Ro, and Scott Holmes. The extensive copyediting required to produce an accurate protocols manual was ably handled by Rebecca Barr, Allen Ranz, Elizabeth Harkins, Lisa Christenson, Connie Parks, Karen Hopkin, Monte Kendrick, and Cathy Lundmark, and electronic illustrations were prepared by Gae Xavier Studios. A basic introductory cell biology text written by the authors of Molecular Biology of the Cell. Two comprehensive and lucid textbooks that convey effectively the synergistic convergence of biochemistry, genetics, structural biology, and traditional cell biology to form modern molecular and cell biology.