Introduction of Flow Cytometry | Flow Cytometry Staining Principles Lesson 3: Exclusionary Antigens

Today, we continue with the third major principle — mutually exclusive antigens can be used with highly interfering fluorochrome combinations. This refers to being mindful of the relationship between antigens during staining. If there is an exclusionary relationship, high interference combinations can be used.

Because flow cytometers have a limited number of channels and multicolor flow experiments require the simultaneous detection of multiple parameters, we often find ourselves using fluorochrome combinations that may cause interference. How can we reduce the effects of this interference on antigen detection? One strategy is to consider the relationship between antigens. When dealing with exclusionary antigens, it's beneficial to prioritize the use of fluorochrome combinations that may have higher interference.

Exclusionary means two antigens will not be expressed simultaneously on the same cell. For example, in CD3⁺ T cells, there's a group of cells expressing CD4 but not CD8, while another group expresses CD8 but not CD4. CD4 and CD8 are considered exclusionary. That is, expressing A excludes the expression of B, and vice versa. During staining, exclusionary antigens allow for fluorochrome leakage.

Figure 1: CD4 and CD8 are mutually exclusive antigens.

Which other antigens demonstrate mutual exclusion? Take CD3 and CD19, for instance. CD3 serves as a marker for T cells, whereas CD19 is indicative of B cells. When concurrently detecting these antigens, employing high-interference combinations is often feasible.

Figure 2: CD3 and CD19 are mutually exclusive antigens.

The marker for NK cells in C57BL/6 mice is CD3-NK1.1+, meaning they express NK1.1 but not CD3. Therefore, CD3 and NK1.1 markers can be detected using high-interference combinations, as shown in the figure 3.

Figure 3: CD3 and NK1.1 are mutually exclusive antigens.

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