Tutorial 4 – Mass Cytometry vs Spectral Flow Cytometry

Panel Design for Spectral Flow and Mass Cytometry: From Principles to Practice

High-parameter cytometry has profoundly transformed our ability to resolve complex immune cell landscapes in a single experiment. Getting the most out of spectral flow and mass cytometry experiments starts at the panel design stage; long before the sample enters the instrument. Poor panel design remains one of the most common sources of data quality issues, from unresolved spectral overlap and autofluorescence interference in spectral flow cytometry, to isotope impurities, oxide formation, and mass spillover in mass cytometry.

This tutorial provides an introduction to panel design principles for both platforms side by side. We will cover the physical and biological sources of signal interference specific to each technology, discuss marker prioritization strategies based on antigen expression levels, and highlight how reagent choice impacts downstream data quality. A particular focus will be placed on the rapidly evolving landscape of AI-assisted panel design tools for spectral flow cytometry, including automated spectral unmixing simulation and fluorochrome compatibility scoring, which are beginning to reshape how panels are built in practice. We will also discuss the importance of selecting appropriate control samples, which are critical to evaluate the performance of a multi-parametric panel.

In the second half of the tutorial, participants will form two groups, one tackling a spectral flow cytometry panel design challenge, the other a mass cytometry challenge, and compete to design an optimized panel under realistic constraints. Results will be discussed and evaluated together, offering insight into the decision-making process and common pitfalls.