Design and Optimization Protocol for High-Dimensional Immunophenotyping Assays using Spectral Flow Cytometry

Technological advances in fluorescence flow cytometry and an ever-expanding understanding of the complexity of the immune system has led to the development of large 20+ flow cytometry panels. Yet, as panel complexity and size increases, so does the difficulty involved in designing a high-quality panel, accessing the instrumentation capable of accommodating large numbers of parameters, and in analysing such high-dimensional data. A recent advancement is spectral flow cytometry, which in contrast to conventional flow cytometry distinguishes the full emission spectrum of each fluorochrome across all lasers, rather than identifying only the peak of emission. Fluorochromes with a similar emission maximum but distinct off-peak signatures can therefore be accommodated within the same flow cytometry panel, allowing greater flexibility in terms of panel design and fluorophore detection. Here, we highlight the specific characteristics regarding spectral flow cytometry and aim to guide users through the process of building, designing and optimising high-dimensional spectral flow cytometry panels using a comprehensive step-by-step protocol. Special considerations are also given for using highly-overlapping dyes and a logical selection process an optimal marker-fluorophore assignment is provided.