Introduction of Flow Cytometry | Flow Cytometry Staining Principles Lesson 4: Co-expressed Antigens

In previous sessions of our flow cytometry course, we covered the three main principles of panel design: matching fluorophore brightness, minimizing spectral overlap, and avoiding co-expressed antigens. Today, we will discuss the fourth principle—avoiding spillover for co-expressed antigens. This principle emphasizes the importance of considering the relationships between antigens when designing a panel. If antigens are co-expressed, it's crucial to avoid fluorescence spillover that could affect the detection of these co-expressed antigens.

The relationships between antigens can be categorized into three types: mutually exclusive, progenitor-progeny, and co-expression.

Mutually Exclusive Relationship: This refers to two antigens that are not expressed simultaneously on the same cell; if antigen A is expressed, antigen B is not, and vice versa. In panel design, it's permissible for mutually exclusive antigens to have fluorescence spillover.

Progenitor-Progeny Relationship: This refers to antigens that are in a hierarchical relationship, where the analysis of the progeny antigen is based on the presence of the progenitor antigen. We will discuss this relationship in more detail in the next session.

Co-expression Relationship: This refers to two antigens that are expressed simultaneously on the same cell. When designing a panel for co-expressed antigens, it is important to choose fluorochromes with minimal spectral overlap to avoid spillover. This session focuses on understanding the co-expression relationship.

What constitutes co-expressed antigens? Take Treg cells as an example: Treg cells express both CD25 and Foxp3. Therefore, CD25 and Foxp3 are considered co-expressed antigens. In this scenario, CD25 and Foxp3 should be labeled with fluorochromes that have minimal spillover, such as APC and PE, since these fluorochromes exhibit minimal interference with each other, allowing for accurate data acquisition.

Figure 1. CD25 and Foxp3 as co-expressed antigens in Treg cells

In multicolor flow cytometry experiments, if the fluorochromes used for two co-expressed markers interfere significantly with each other, it can affect gating accuracy and compromise the experiment's results. Although compensation can be adjusted to reduce the impact of spectral overlap, it cannot eliminate the reduced fluorescence sensitivity in the affected channel during subsequent data analysis.

Figure 2. Effects of Fluorescence Interference

What happens if there is significant fluorescence interference between co-expressed antigens? The result might be similar to the situation shown in the right image above. Despite compensation adjustments, the proportion of target cells obtained may differ from the actual value.

In the next session of our flow cytometry lesson, we will analyze the progenitor-progeny relationship in panel design principles. Stay tuned for more insights in the Flow Cytometry Guide column!