1. Use the best combination of fluorochromes.

Caution is needed when constructing a spectral flow cytometry panel, even when faced with the possibility of using fluorochrome combinations that would not be feasible in conventional flow cytometry.

In addition to paying attention to the principles of matching fluorochrome brightness to antigen expression levels and selecting appropriate fluorochromes for co-expressing antigens, spectral flow cytometry also requires choosing the best combination of fluorochromes, considering their cosine similarity (CS, more frequently referred to as similarity index), spillover-spreading error (SSE), and unmixing-spreading error (SE).

When your panel shows more fluorochromes with discrepant cosine similarity, as well as lower spillover-spreading error (SSE) and unmixing-spreading error values, the unmixing calculation will be better, the spillover (fluorescence overlap) will be lower, and thus the resolution of your labeling will be higher. In other words, the more distinct the spectral signatures of each fluorochrome present in the panel and the more correctly they are correlated with the characteristics of the antigens, the easier it will be to identify them in the unmixing process and the more clearly the positive and negative populations will be separated from each other. The best fluorochromes have lower cosine similarity, reduced spillover-spreading matrix values, and lower unmixing-spreading error.

Over the next two days, we will gain a better understanding of what cosine similarity (CS), spillover-spreading error (SSE), and unmixing-spreading error (SE) are.

Count on CYTO-flowing for the review of your flow cytometry panels.

doi: 10.1002/cyto.a.24841

doi: 10.1101/2025.04.17.649396

doi: 10.1002/cyto.a.22251