干细胞之家www.stemcell8.cn ←点击进入 METHODSMETHODS IN IN MOLECULAR BIOLOGYTMTM Volume 263 FlowFlow CytometryCytometry ProtocolsProtocols SSECONDECOND EEDITIONDITION Edited by Teresa S. Hawley Robert G. Hawley 干细胞之家www.stemcell8.cn ←点击进入 1 Flow Cytometry An Introduction Alice L. Givan Summary A flow cytometer is an instrument that illuminates cells (or other particles) as they flow indi- vidually in front of a light source and then detects and correlates the signals from those cells that result from the illumination. In this chapter, each of the aspects of that definition will be described: the characteristics of cells suitable for flow cytometry, methods to illuminate cells, the use of fluidics to guide the cells individually past the illuminating beam, the types of signals emitted by the cells and the detection of those signals, the conversion of light signals to digital data, and the use of computers to correlate and analyze the data after they are stored in a data file. The final section of the chapter will discuss the use of a flow cytometer to sort cells. This chap- ter can be read as a brief, self-contained survey. It can also be read as a gateway with signposts into the field. Other chapters in this book will provide more details, more references, and even some controversy about specific topics. Key Words Flow cytometry, fluidics, fluorescence, laser. 1. Introduction An introductory chapter on flow cytometry must first confront the difficulty of defining a flow cytometer. The instrument described by Andrew Moldavan in 1934 (1) is generally acknowledged to be an early prototype. Although it may never have been built, in design it looked like a microscope but provided a cap- illary tube on the stage so that cells could be individually illuminated as they flowed in single file in front of the light emitted through the objective. The signals coming from the cells could then be analyzed by a photodetector attached in the position of the microscope eyepiece. Following work by Coul- From: Methods in Molecular Biology: Flow Cytometry Protocols, 2nd ed. Edited by: T. S. Hawley and R. G. Hawley © Humana Press Inc., Totowa, NJ 1 干细胞之家www.stemcell8.cn ←点击进入 2Givan ter and others in the next decades to develop instruments to count particles in suspension (see refs. 2–5),adesign was implemented by Kamentsky and Melamed in 1965 and 1967 (6,7) to produce a microscope-based flow cytome- ter for detecting light signals distinguishing the abnormal cells in a cervical sample. In the years after publication of the Kamentsky papers, work by Fulwyler, Dittrich and Göhde, Van Dilla, and Herzenberg (see refs. 8–11) led to significant changes in overall design, resulting in a cytometer that was largely similar to today’s cytometers. Like today’s cytometers, a flow cytometer in 1969 did not resemble a microscope in any way but was still based on Moldavan’s prototype and on the Kamentsky instrument in that it illuminated cells as they progressed in single file in front of a beam of light and it used photodetectors to detect the signals that came from the cells (see Shapiro [12] and Melamed [13,14] for more complete discussions of this historical development). Even today, our definition of a flow cytometer involves an instrument that illuminates cells as they flow individually in front of a light source and then detects and cor- relates the signals from those cells that result from the illumination. In this chapter, each of the aspects in that definition are described: the cells, methods to illuminate the cells, the use of fluidics to make sure that the cells flow individually past the illuminating beam, the use of detectors to mea- sure the signals coming from the cells, and the use of computers to correlate the signals after they are stored in data files. As an introduction, this chapter can be read as a brief survey; it can also be read as a gateway with signposts into the field. Other chapters in this book (and in other books [e.g., refs. 12,15–24) provide more details, more references, and even some controversy concerning specific topics. 2. Cells (or Particles or Events) Before discussing “cells,” we need to qualify even that basic word. “Cytome- ter” is derived from two Greek words, “κντοζ”, meaning container, receptacle, or body (taken in modern formations to mean cell), and “µετρον”, meaning measure. Cytometers today, however, often measure things other than cells. “Par- ticle” can be used as a more general term for any of the objects flowing through a flow cytometer. “Event” is a term that is used to indicate anything that has been interpreted by the instrument, correctly or incorrectly, to be a single parti- cle. There are subtleties here; for example, if the cytometer is not quick enough, two particles close together may actually be detected as one event. Because most of the particles sent through cytometers and detected as events are, in fact, single cells, those words are used here somewhat interchangeably. Because flow cytometry is a technique for the analysis of individual parti- cles, a flow cytometrist must begin by obtaining a suspension of particles. His- torically, the particles analyzed by flow cytometry were often cells from the 干细胞之家www.stemcell8.cn ←点击进入 Flow Cytometry: An Introduction 3 blood; these are ideally suited for this technique because they exist as single cells and require no manipulation before cytometric analysis. Cultured cells or cell lines have also been suitable, although adherent cells require some treat- ment to remove them from the surface on which they are grown. More recently, bacteria (25,26),sperm (27,28), and plankton (29) have been analyzed. Flow techniques have also been used to analyze individual particles that are not cells at all (e.g., viruses [30], nuclei [31], chromosomes [32],DNA fragments [33], and latex beads [34]). In addition, cells that do not occur as single particles can be made suitable for flow cytometric analysis by the use of mechanical disruption or enzymatic digestion; tissues can be disaggregated into individual cells and these can be run through a flow cytometer. The advantage of a method that analyzes single cells is that cells can be scanned at a rapid rate (500 to >5000 per second) and the individual characteristics of a large number of cells can be enumerated, correlated, and summarized. The disadvantage of a single- cell technique is that cells that do not occur as individual particles will need to be disaggregated; when tissues are disaggregated for analysis, some of the char- acteristics of the individual cells can be altered and all information about tissue architecture and cell distribution is lost. In flow cytometry, because particles flow in a narrow stream in front of a narrow beam of light, there are size restrictions. In general, cells or particles must fall between approx 1 µm and approx 30 µm in diameter. Special cytome- ters may have the increased sensitivity to handle smaller particles (such as DNA fragments [33] or small bacteria [35]) or may have the generous fluidics to handle larger particles (such as plant cells [36]). But ordinary cytometers will, on the one hand, not be sensitive enough to detect signals from very small par- ticles and will, on the other hand, become obstructed with very large particles. Particles for flow cytometry should be suspended in buffer at a concentration of about 5 × 105 to 5 × 106/mL. In this suspension, they will flow through the cytometer mostly one by one. The light emitted from each particle will be detected and stored in a data file for subsequent analysis. In terms of the emit- ted light, particles will scatter light and this scattered light can be detected. Some of the emitted light is not scattered light, but is fluorescence. Many par- ticles (notably phytoplankton) have natural background (auto-) fluorescence and this can be detected by the cytometer. In most cases, particles without intrinsically interesting autofluorescence will have been stained with fluorescent dyes during preparation to make nonfluorescent compounds “visible” to the cytometer. A fluorescent dye is one that absorbs light of certain specific colors and then emits light of a different color (usually of a longer wavelength). The fluorescent dyes may be conjugated to antibodies and, in this case, the fluo- rescence of a cell will be a readout for the amount of protein/antigen (on the cell surface or in the cytoplasm or nucleus) to which the antibody has bound. 干细胞之家www.stemcell8.cn ←点击进入 4Givan Some fluorochrome-conjugated molecules can be used to indicate apoptosis (37). Alternatively, the dye itself may fluoresce when it is bound to a cellular component. Staining with DNA-sensitive fluorochromes can be used, for exam- ple, to look at multiploidy in mixtures of malignant and normal cells (31); in conjunction with mathematical algorithms, to study the proportion of cells in different stages of the cell cycle (38); and in restriction-enzyme-digested mate- rial, to type bacteria according to the size of their fragmented DNA (39). There are other fluorochromes that fluoresce differently in relation to the concentra- tion of calcium ions (40) or protons (41,42) in the cytoplasm or to the poten- tial gradient across a cell or organelle membrane (43). In these cases, the fluorescence of the cell may indicate the response of that cell to stimulation. Other dyes can be used to stain cells in such a way that the dye is partitioned between daughter cells on cell division; the fluorescence intensity of the cells will reveal the number of divisions that have occurred (44). Chapters in this book provide detailed information about fluorochromes and their use. In addi- tion, the Molecular Probes (Eugene, OR) handbook by Richard Haugland is an excellent, if occasionally overwhelming, source of information about fluor- escent molecules.