Unraveling Immune Cell Diversity: Immunophenotyping via Flow Cytometry

The History of Flow Cytometry

In 1934, Andrew Moldavan tried to use a photodetector to analyze a single cell flowing through a capillary and proposed the design concept that is widely regarded as the earliest prototype of flow cytometry. Since then, flow cytometry has undergone early evolution, and it developed rapidly in the 1970s. In 1967, Van Dilla and Los Alamos used a laminar flow chamber and an argon laser to develop a flow cytometer in which the liquid flow beam, the illumination optical axis, and the detection system were perpendicular to each other. This became the basis for a variety of current flow cytometers. In 1975, George Kohler and Cesar Milstein proposed monoclonal antibody technology. On this basis, biological researchers began to manufacture monoclonal antibodies against leukocytes, obtained a series of clones capable of producing monoclonal antibodies targeting leukocyte surface differentiation antigens and labeled antibodies with fluorescent dyes, which provided a large number of antibodies for flow cytometry research.

Flow cytometry has the characteristics of fast and high throughput. Compared with WB, it can analyze the expression of multiple targets in the same sample in a shorter time. Combined with cell phenotype markers, specific cell groups and subpopulations can be analyzed. Therefore, it has incomparable advantages in immunology research.

Immunophenotyping and Flow Cytometry in Cell Analysis

The immune system includes immune organs, immune cells, and immune molecules, among which immune cells are the main executors of the specific functions of the immune system. According to the type of immune response, it is divided into innate immune cells and adaptive immune response cells. Immune cell markers are widely used in flow cytometry to identify and differentiate different types of immune cells. The cells involved in the innate immune response include macrophages, dendritic cells, NK cells, NKT cells, innate lymphoid cells, granulocytes, mast cells, etc. The cells involved in the adaptive immune response are mainly T and B cells expressing specific antigen recognition receptors (TCR/BCR).

Immunophenotyping refers to analyzing heterogeneous cell populations to identify the presence and proportion of various target cell populations. The process uses antibodies for identification that specifically detect antigens expressed by these cells, known as markers. These markers are often functional membrane proteins involved in cellular communication, adhesion, or metabolism. Immunophenotyping by flow cytometry is the method of choice for identifying and classifying cells in complex cell populations, for example, analyzing immune cells in a blood sample. The technique can be applied in both basic research and clinical laboratories.

Cell markers are an effective way to identify specific cell populations, however, often more than one cell type expresses these markers. Therefore, for the immunophenotyping of cells, the flow cytometry staining method needs to use more than two kinds of antibodies at the same time. Specific cell populations are identified and quantified by assessing the specific expression of cell markers using multiple antibodies simultaneously, each conjugated to a different fluorochrome. Many immune cell markers are CD markers that are commonly used in flow cytometry to detect specific immune cell populations and subpopulations. The following are common CD markers used for immunophenotyping.