Immunophenotyping is the analysis of heterogeneous populations of cells for the purpose of identifying the presence and proportions of the various populations of interest. Antibodies are used to identify cells by detecting specific antigens expressed by these cells, which are known as markers. These markers are usually functional membrane proteins involved in cell communication, adhesion, or metabolism. Immunophenotyping using flow cytometry has become the method of choice in identifying and sorting cells within complex populations, for example the analysis of immune cells in a blood sample. Applications of this technology are used both in basic research and clinical laboratories.
Cell markers are a very useful way to identify a specific cell population. However, they will often be expressed on more than one cell type. Therefore, flow cytometry staining strategies have led to methods for immunophenotyping cells with two or more antibodies simultaneously. By evaluating the unique repertoire of cell markers using several antibodies together, each coupled with a different fluorochromes, a given cell population can be identified and quantified. Many immunological cell markers are CD markers and these are commonly used for detection in flow cytometry of specific immune cell populations and subpopulations.
Immune cell differentiation and CD markers
Lymphoid cell differentiation CD markers
T cells, B cells, Nk cells and plasmacytoid dendritic cells.
Myeloid cell differentiation CD cell markers
Monocytes, macrophages, granulocytes, platelets, myeloid dendritic cells.
Flow cytometry is the measurement (-metry) of cellular (cyto-) properties as they are moving in a fluid stream (flow), past a stationary set of detectors. Latest developments go a step further and allow cells of different subtypes to be sorted and collected for further analysis. It is capable of rapid, quantitative, multi-parameter analysis of heterogeneous cell populations on a cell-by-cell basis (single cell analysis).
Flow cytometry protocols for cell membrane marker protein staining
As the number of antibodies used for phenotyping increases so does the complexity caused by the overlapping spectra of the fluorochromes. Controls must also be evaluated alongside the experimental samples to insure the data is collected and interpreted correctly.