A practical approach to multicolor flow cytometry for immunophenotyping

Through a series of novel developments in flow cytometry hardware, software , and dye-chemistry it is now possible to simultaneously measure up to II d istinct fluorescences and two scattered light parameters on each cell. Such advanced multicolor systems have a number of advantages over current two- and three-color flow cytometric measurements. They provide a large amount o f novel information for each sample studied, an exquisitely accurate quanti tation of even rare cell populations, and allow identification and characte rization of novel cell subsets. In particular, this technology is proving c rucial to identifying functionally homogeneous subsets of cells within the enormously complex immune system; such identification and enumeration is im portant for understanding disease pathogenesis. However, multicolor flow cy tometry comes with a new and sometimes difficult set of technical problems that must be overcome by users to derive meaningful results. In this manusc ript, we describe the basic aspects of multicolor flow cytometry, including the technical hurdles and artefacts that may occur, and provide some sugge stions for how to best overcome these hurdles. While inspired by the Ii-col or technology that we currently use, these principles apply to all flow cyt ometric experiments in which more than one fluorescent dye is used. (C) 200 0 Elsevier Science B.V. All rights reserved.