When preparing a sample for flow cytometric analysis, reagents that contain MoAbs against the antigens, or clusters of differentiation (CD), of interest are used to help identify populations of cells. Clusters of differentiation are specific antigens that aid in the identification of white cells, which are the targets of commercially prepared MoAbs in staining kits. These MoAbs are grouped according to the antigens that they recognize into these clusters of differentiation. For example, the key CD markers for B lymphcytes are CD19 and CD20, while the key CD markers for T lymphocytes are CD3, CD4, and CD8. Each white blood cell has specific CD markers that can be used for identification purposes in flow cytometry.
To expand on this concept further, cluster of differentiation 3 (CD3), for example, binds to all mature T cells (helper and suppressor) while CD4 binds to helper T cells only. Using this scenario, when a sample is stained with the fluorochromes, fluorescein isothiocyanate (FITC) and phycoerythrin (PE):
- Suppressor T cells (CD3-bound) will fluoresce orange, but not green
- Helper T cells (CD3- and CD4-bound) will fluoresce both orange and green
- B cells, monocytes, and neutrophils should not express either antigen; thus they should not emit a fluorescent signal when they intersect the laser.