attenuate any remaining scattered laser light, leaving only the light emitted by the cell-bound fluorochromes. This emitted light is then deflected to the FLI PMT, using 550-nm DL and 525-nm BP filters. Thus, the FLl PMT captures a narrow bandwidth of light centering on 525 nm (green fluorescence emitted, e.g., by the dye FITC). In the three-color instrument shown here, this process is repeated two more times, with the FL2 and FL3 PMTs capturing light of progressively longer wavelengths, emitted by additional fluorochromes. Commercially available cytometers with multiple lasers and photodetectors have been used to measure as many as 14 fluorochromes simultaneously, using the same principles (Modified from Beckman Coulter Manual. COULTER EPICS XL/XL-MCL FLOW CYTOMETER, PN 4237340AI-I, 6 April 1998, Elite XL Cytometer.). In the simplest flow cytometers, light is supplied by a single air-cooled argon or diode laser that emits blue light at a wavelength of 488 nm. Ultraviolet, violet, green, yellow, and red lasers are becoming increasingly common as the list of available fluorochromes grows and multiparameter flow cytometry is more widely adopted. The most commonly used fluorochromes include the old standbys fluorescein T understanding Clinical flow Cytornetry lU5 isot hiocyanate (FITC), phycoerythrin (PE) and the PE-tandclll dyes (PE-Texas red, PE-Cy5, PE-Cy5.5, and PE-Cy7), pcridinin chlorophyll protein (PerCP), allophycocyanin (APC), and the APC tandems: APC-Cy5.5 and APC-Cy7. The tandem dyes are of particular importance because they allow a single laser to excite as Illany as six separate fluorochromes (e.g., FlTC. PE, and the PE-tandems can all be excited by a 488-nlll laser). PE is a large fluorescent protein that can be excited at 488 nm (but is more efficiently excited by a green or yellow laser), and it emits at 578 nlll. It can be decorated with small dye molecules such as Texas red, Cy5, Cy5.5, or Cy7, which are not excited by themselves at 488 nm. Known asfree energy transfer in physics, the excited PE, instead of emitting a photon at a higher wavelength, directly excites the coupled dye, which then emits light at its characteristic wavelength. Several different PE-tandelll dyes with increasingly longer Stokes shifts can be used simultaneously with a single blue or green laser capable of exciting PE. The cyanine and Alexa dye families represent a new generation of designer dyes, which are specifically engineered for their excitation and emission characteristics. Increasingly, antibodies conjugated to a variety of fluorochromes are commercially available. For the direct staining procedure, a cell suspension is initially incubated with one or more dye-coupled monoclonal antibodies directed against the determinants of interest. In the indirect assay, an unlabeled antibody against one target molecule is used to stain the cells. The presence of this antibody is detected by a second labeled antibody. For example, T cells can be identified in human peripheral blood by staining with an unlabeled murine antibody directed against CD3. After washing away the unbound antibody, a fluorescence-labeled goat antimouse immunoglobulin antibody can be used to detect the presence of anti-CD3 on the human T cells. Indirect staining can be used in concert with direct staining, provided the indirect staining is performed first, and all free antimouse immunoglobulin-binding sites are blocked (with mouse serum or purified mouse IgG) before adding the directly conjugated murine antibodies. A relatively new development in which small quantities of monoclonal antibody can be labeled with the antigen-specific portion (FAB) of an antiglobulin conjugated to a dye of choice, provides additional flexibility [21]. Finally, antibodies that are directly labeled with biotin can be purchased. Biotin is not fluorescent by itself, but binds to the tetrameric molecule avidin or streptavidin with an unusually high affinity (hence it's name), which in turn can be conjugated to the dye of choice. This provides versatility, since a biotin-conjugated antibody can be used with your choice of avidin-conjugated fluorochromes, and conversely, the same avidin-conjugated fluorochrome can be used with an array of different biotinylated antibodies. In