Best practices in high-dimensional spectral panel design: a step-by-step guide to success

Recent advances in cytometry instrumentation and reagents have increased the maximum possible dimensionality of a fluorescence-based flow cytometry experiment up to 50 different markers. However, without appropriate guidance designing these high-dimensional panels can be time-consuming and frustrating.
In this tutorial, we outline systematic workflows to design any panel from 10-50 parameters on conventional and spectral cytometers, while minimizing wet-lab trial and error. After covering some theoretical and practical basics that are relevant for panel design, we will use an interactive format to discuss some typical caveats and problems. Specifically, we will:
• Discuss the relevance of spillover spreading error (SE) for panel design
• Outline advantages and limitations of the similarity and complexity index
• Introduce a novel type of spreading error specific to spectral cytometry
• Summarize a step-by-step workflow for panel design
The overarching goal of this tutorial is to provide all participants with the knowledge and tools they need to successfully design and troubleshoot their polychromatic cytometry experiments irrespective of the platform used.