CD Markers are especially useful for identification of leukocyte population using flow cytometry. Our resource pages General flow cytometry protocol and What is flow cytometry? provide a short introduction into flow cytometry. The great advantage of flow cytometry is that it allows for the simultaneous detection of several markers on a single cells at the very same time. With a modern flow cytometer 8-10 different colors can easily be measured in one sample, the most advanced cytometers can even measure up to 18 channels at once.
Table of the most common CD markers for flow cytometry
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|Natural Killer Cells||CD56||CD335||(CD56)||(CD56)||(CD56)||(CD56)|
|Monocytes / Macrophages||CD14|
|(Macrophage marker)||(Macrophage marker)||(Macrophage marker)||(Macrophage marker)||(CD33)|
|Hematopoetic stem cells||CD34||CD34||(CD34)||(CD34)||(CD34)|
Use of CD-Marker for the determination of leukocyte populations
When using flow cytometry to determine certain leukocyte population usually a combination of several CD markers is used to determine a subpopulation, since one single marker usually falls short of defining the entire population. Usually research generate gating trees when evaluating flow cytometry experiments.
A very illustrative example is the distribution of CD8. While CD8 is the most prominent marker and namesake for CD8 positive (CD8+) cytotoxic T cells, it is also expressed by natural killer cells (NK-cells) and dentritic cells (DCs). If all CD8+ cells were gated from a leukocyte population of a blood sample the gated population would consist of CD8+ T cells, NK cells and DCs.
In the following experiment the ratio of CD4+ T helper vs. CD8+ cytotoxic T cells was determined. First all CD45+ cells (general leukocyte marker) of the peripheral blood sample were gated (Gate 1).
In the next panel all CD45+ cells of Gate 1 were blotted for their expression of CD3. CD3 is the most common T cell marker which is only expressed by T cells and not by other CD8+ cells such as NK cells or DC. By gating for CD3+ cells it is guaranteed that no CD3- but CD8+ cells (e.g. NK cells or DCs) are counted as CD8+ T cells.
The CD3+ T cell population can be further differentiated into cytotoxic CD8+ T cells and into CD4+ T helper cells as shown in panel2 where the CD3+ cells of Gate 2 were blotted for their expression CD4 and CD8. CD4+ CD8- T helper cells are found in the upper left quadrant and CD4- CD8+ cytotoxic T cells are shown in lower right quadrant of panel 3.
If needed a researcher interested in different T helper Population such as TH1, TH2, TH9, TH17 and Treg would stain for the respective extra or intracellular markers of those subpopulation.