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).