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High-Dimensional Fluorescence Cytometry
Thomas Myles Ashhurst1,2,3,4, Adrian Lloyd Smith2,3, Nicholas Jonathan Cole King1,2,3,4,5
1Viral Immunopathology Laboratory, Discipline of Pathology, School of Medical Sciences, Sydney Medical School, The University of Sydney, Sydney, Australia.
Insights
This review guides users in building and analyzing high-dimensional fluorescence flow cytometry panels for immune cell analysis. It covers essential principles for constructing quality panels and interpreting complex datasets.
Area of Science:
- Immunology
- Cell Biology
- Biotechnology
Background:
- The immune system comprises diverse cells expressing various proteins.
- Flow cytometry with fluorophore-conjugated antibodies enables cell analysis.
- Increasing immune system complexity drives demand for high-dimensional flow cytometry panels.
Purpose of the Study:
- To review principles for constructing high-dimensional flow cytometry panels.
- To guide users in building and analyzing quality panels.
- To address challenges in high-dimensional data generation and analysis.
Main Methods:
- Discussion of key principles for panel design.
- Guidance on optimizing panel construction and data analysis.
- Consideration of factors like fluorophore properties and antibody selection.
Main Results:
- Identification of critical factors for high-dimensional panel construction.
- Strategies for overcoming challenges in analyzing complex flow cytometry data.
- A comprehensive approach to building and analyzing robust panels.
Conclusions:
- Effective high-dimensional panel design is crucial for accurate immune cell analysis.
- Understanding principles of fluorophore selection, optimization, and data analysis is essential.
- This review provides a framework for successful high-dimensional flow cytometry studies.
Abstract:
The immune system consists of a complex network of cells, all expressing a wide range of surface and/or intracellular proteins. Using flow cytometry, these cells can be analyzed by labeling with fluorophore-conjugated antibodies. The recent expansion of fluorescence flow cytometry technology, in conjunction with the ever-expanding understanding of the complexity of the immune system, has led to the generation of larger high-dimensional fluorescence flow cytometry panels. However, as panel size and complexity increases, so too does the difficulty involved in constructing high-quality panels, in addition to the challenges of analyzing such high-dimensional datasets. As such, this unit seeks to review the key principles involved in building high-dimensional panels, as well as to guide users through the process of building and analyzing quality panels. Here, cytometer configuration, fluorophore brightness, spreading error, antigen density, choosing the best conjugates, titration, optimization, and data analysis will all be addressed. © 2017 by John Wiley & Sons, Inc.
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