Monoclonal antibodies (MoAbs) are labeled with fluorescent particles. These MoAbs will bind specifically to corresponding cell surface antigens.
Since blood cells may have multiple surface antigens, a single cell can bind to more than one type of labeled MoAb; depending upon the MoAbs that are used.
Different combinations of antibodies can be used within the same staining tube, as long as the fluorochromes will emit signals that can be separated from one another; keeping in mind that compensation for spectral overlap may be necessary. For example, if two separate monoclonal antibodies are labeled with a red and orange fluorochrome respectively, it is important to compensate for the closeness of the two colors on the color spectrum and to make sure that the red-tagged MoAb signal is captured as red and the orange-tagged MoAb signal is captured as orange (and red isn't registered as orange and vice versa).
In the image shown on the right, the cells were stained with MoAb-green fluorochrome and MoAb-orange fluorochrome. The cell that is illustrated expresses both antigens and the antibodies that are bound to the respective antigens.
The laser beam excites the fluorochromes and fluorescent signals are emitted. The signals are captured and separated from other fluorescent signals by filters and mirrors.
