The significance of circling lymphocytes through CD45 and SSC

In the aritle "Human peripheral Blood regulatory T cell identification Scheme", we used CD45 to distinguish lymphocytes. There are doubts that: in human peripheral blood, all the white blood cells express CD45 on the surface, and there is no need to add a CD45 antibody to the peripheral blood sample. With FSC/SSC, lymphocytes can be circled directly.

Is CD45 really dispensable when doing immune cell typing? Let's talk about the characteristics of CD45 and the significance of circling lymphocytes by CD45 and SSC-H.

CD45 is composed of a class of transmembrane proteins with similar structure and large molecular weight, and widely exists on the surface of leukocytes. CD45 molecule is expressed on all leukocytes and is a key molecule in cell membrane signal transduction. It plays an important role in the development and maturation of lymphocytes, functional regulation and signal transmission, and its distribution can be used as a classification marker for some T cell subsets.

Figure 1. Comparison of CD45/SSC-H and FSC-H/SSC-H cell clusters

As shown on the left, with CD45 and SSC-H, lymphocytes can be circled very quickly.

As shown in the figure on the right, when FSC-H and SSC-H are used to determine the distribution of cell populations, if CD45 is not stained, and sometimes the effect of red blood cell lysis is not good, the lymphocyte population is easy to be unclear with red blood cells and cell fragments, and monocytes are easy to be mixed, which is not conducive to the determination of the location of the ring gate. Through the CD45/SSC ring gate, purer circled lymphocytes can be obtained.

The lymphocyte population usually showed CD45high and SSClow. Compared with lymphocytes, CD45low and SSCmid is monocyte. Both CD45low and SSClow is basophils. At the same time, CD45 is present in each sample tube of the three-color and four-color antibody combination scheme, and it also play a role in monitoring and evaluating the variability between the sample tubes while setting a gate for lymphocyte populations.

Figure 2. Scatter plots of CD45 and SSC cells in peripheral blood

So, under what circumstances must CD45 be dyed?

For example, when the distribution of peripheral blood samples of some patients is quite different from that of normal samples, lymphocytes must be separated by CD45.

In addition, the tumor samples that have been studied more recently must also be stained with CD45 when detecting immune cell clusters. Because most of the single-cell suspensions treated by tumor tissues are tumor cells and histiocytes, and the content of immune cells is generally small, and if CD45 is not stained, the results cannot be analyzed.

To sum up, when the sample is poorly processed or the sample type is relatively special lead to the clear immune cell morphology cannot be distinguished by FSC/SSC, in these cases, the combination of CD45 and SSC is crucial and necessary.