Introduction of Flow Cytometry | Flow Cytometry Staining Principles Lesson 5: Progenitor and Descendant Antigens

The previous articles introduced four key principles: strength matching, fluorescence interference, mutually exclusive antigens, and co-expressed antigens and this article will introduce the fifth rule of multicolor flow cytometry panel design: allowing fluorescence spillover from descendant to progenitor, but not the reverse. This principle means that if the antigens being stained have a hierarchical relationship, fluorescence spillover from the descendant antigen to the progenitor antigen is permissible, whereas spillover from the progenitor antigen to the descendant antigen should be avoided.

In this context, progenitor and descendant refer to the hierarchical relationship between antigens, where the analysis of the descendant antigen is based on the progenitor antigen. For example, within CD3+ T cells, there is a subset that expresses CD4. Here, CD3 is the progenitor and CD4 is the descendant. Thus, in panel design, it is acceptable for CD4 to have fluorescence spillover into CD3, but CD3 should not have spillover into CD4.

In multicolor flow cytometry experiments, since cells expressing CD4 also express CD3, any fluorescence spillover from CD4 to CD3 will not affect the detection of CD3 (as shown in Figure 1). However, if CD3 has fluorescence spillover into CD4, the spillover fluorescence from CD3 can interfere with the detection of CD4 (as shown in Figure 2).

Figure 1. Results of CD4-FITC and CD3-PE dual staining

Figure 2. Results of CD3-FITC and CD4-PE dual staining

For markers like CD3 and CD4, which have distinct population separations, the impact of background signal interference might be minimal. However, for markers with low expression or continuous expression, background interference due to fluorescence spillover can be significant.

Take Th1 cells as an example. Th1 cells require the detection of CD4 and IFN-γ expression, where CD4 is the progenitor and IFN-γ is the descendant. Because the PE/Cyanine7 channel has substantial fluorescence spillover into the PE channel, IFN-γ can be paired with PE/Cyanine7 and CD4 with PE. In this case, IFN-γ is in a non-interfering channel, making it easier to adjust compensation, which is beneficial for data analysis.

With this, we have concluded the principles of flow cytometry panel design. In the next issue of our flow cytometry series, we will provide a summary. Stay tuned!