CYTO 2025 Tutorials: The road to successful high-parameter spectral cytometry experiments: guidelines for control optimization, handling of autofluorescence and quality control

The road to successful high-parameter spectral cytometry experiments: guidelines for control optimization, handling of autofluorescence and quality control
Andrew Konency - Vaccine and Infectious Disease Division, Fred Hutch Cancer Center
Oliver Burton, PhD - Department of Pathology, University of Cambridge
Maria Jaimes, MD - Cytek Biosciences
Joanne Lannigan, MS - Flow Cytometry Support Services, LLC
Florian Mair, PhD - Managing Director, Flow Cytometry Core Facility, ETH Zurich

Spectral flow cytometry has expanded the boundaries of high-dimensional analysis, enabling deeper biological insights than ever before. However, the ever-increasing plexity of multiparameter panels introduces unique challenges in experiment design, quality control (QC), and data interpretation. To fully optimize an assay, distinct considerations are required to prepare appropriate control samples, including unstained controls, as well as a rigorous quality assessment strategy.

In this tutorial we will discuss common caveats and strategies that are needed for the successful setup of a high-dimensional spectral cytometry experiment. In addition to an overview of current best practices and practical tips and tricks, attendees will gain insight into: - Optimization of experimental procedures and single-stained controls - How to best manage autofluorescence - Effective use of control samples for multicolor data QC

By showing real-world examples, this tutorial will highlight both emerging solutions and well-documented approached to overcome typical challenges encountered with highly complex flow cytometry assays and will provide participants with actionable strategies to improve their experimental designs and analysis workflows. By the end of the session, attendees will be equipped with the knowledge to anticipate, troubleshoot and mitigate common pitfalls related to controls and autofluorescence, that will contribute to the generation of reproducible and high-quality spectral cytometry data.

CMLE Credit: 1.0