Flow cytometry, also called flow microfluorometry or flow cytofluorometry, employs instrumentation that scans single cells flowing past excitation sources in a liquid medium. The technology is unique in its ability to provide rapid, quantitative, multiparameter analyses on single living (or dead) cells. Measurement of visible and fluorescent light emission allows quantitation of antigenic, biochemical, and biophysical characteristics of individual cells. Flow cytometry technology can also separate distinct subpopulations of cells on the basis of these measured characteristics, usually by electrostatic deflection. This separation technology is called electronic cell sorting.
Flow cytometry has two general applications— quantitative analysis and cell separation. Cell separation is dependent upon the analytical capabilities of flow cytometry, in that cells of interest must be identified analytically in order to be separated. A graphical representation of flow cytometry analytical measurements made on individual cells is known as a histogram. Data plotted on a histogram include the number of cells and values of one or more measurements made on individual cells.
Performing flow cytometry experiments generally involves three distinct, interdependent phases. First is the pre–flow cytometry phase which involves reagent preparation, cell preparation, protocol design, and staining of cells with the fluorescent reagents. Second is the flow cytometry phase which involves processing the stained cells using flow cytometry instrumentation and collecting data for one or more measurements (parameters) made on each individual cell. Finally, the analysis phase involves analyzing the collected data. These three phases may all be performed by a single individual during the course of one day. Alternatively, each of the three phases may be performed by different persons and at different points in time. For example, one person might stain the cells and deliver them to a specialized flow cytometry facility for processing the next day. The data might then be analyzed by a third individual at a later time. Details on protocols for each of these phases of flow cytometry experiments are found throughout this chapter. This introductory unit provides an overview of terminology and techniques unique to flow cytometry and is divided into two sections. The first section discusses technical aspects of flow cytometry which apply primarily to the instrumentation- oriented flow cytometry phase. The second section discusses electronic cell separation using flow cytometry. The flow cytometry phase for specific instruments is presented in UNIT 5.4.
UNIT 5.2 provides techniques for analysis of flow cytometry data, with emphasis on the analysis of immunofluorescence data. The pre– flow cytometry phase, including cell preparation and staining as well as fluorochrome-conjugated reagent preparation, is presented in UNIT 5.3. UNITS 5.2-5.4 emphasize cell surface immunofluorescence applications in flow cytometry, while UNIT 21.4 presents an alternative nonfluorescent technique for surface antigen analysis using conventional microscopy. Additional specific applications of flow cytometry in immunobiology are presented in UNITS 5.5-5.7. UNIT 5.5 (intracellular ions) and UNIT 5.7 (DNA) focus on measurement of intracellular materials using flow cytometry. UNIT 5.6 presents a protocol which uses flow cytometry to measure cell:cell conjugates. TERMINOLOGY AND
