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 are recognized by a group of similar MoAbs. CDs are detected by commercially prepared MoAbs in staining kits to aid in identifying white blood cell populations. For example, the key CD markers for B lymphocytes are CD19 and CD20, while those 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, c
luster of differentiation 3 (CD3) binds to all mature T cells (helper and cytotoxic) while CD4 binds to helper T cells. Using this scenario, when a sample is stained with the fluorochromes, fluorescein isothiocyanate (FITC) and phycoerythrin (PE):
- Cytotoxic 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.