A practical approach to multicolor flow cytometry for immunophenotyping

J Immunol Methods. 2000 Sep 21;243(1-2):77-97. doi: 10.1016/s0022-1759(00)00229-5.


Through a series of novel developments in flow cytometry hardware, software, and dye-chemistry it is now possible to simultaneously measure up to 11 distinct 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 of novel information for each sample studied, an exquisitely accurate quantitation of even rare cell populations, and allow identification and characterization of novel cell subsets. In particular, this technology is proving crucial to identifying functionally homogeneous subsets of cells within the enormously complex immune system; such identification and enumeration is important for understanding disease pathogenesis. However, multicolor flow cytometry comes with a new and sometimes difficult set of technical problems that must be overcome by users to derive meaningful results. In this manuscript, we describe the basic aspects of multicolor flow cytometry, including the technical hurdles and artefacts that may occur, and provide some suggestions for how to best overcome these hurdles. While inspired by the 11-color technology that we currently use, these principles apply to all flow cytometric experiments in which more than one fluorescent dye is used.

Publication types

  • Review

MeSH terms

  • Antibodies, Monoclonal / immunology
  • Flow Cytometry / instrumentation
  • Flow Cytometry / methods*
  • Fluorescent Dyes / metabolism
  • Humans
  • Immunophenotyping / methods*
  • Lasers
  • Quality Control


  • Antibodies, Monoclonal
  • Fluorescent Dyes