High-Dimensional Fluorescence Cytometry

• Published in: Current protocols in immunology Vol. 119; p. 5.8.1
• Main Authors: Ashhurst, Thomas Myles, Smith, Adrian Lloyd, King, Nicholas Jonathan Cole
• Format: Journal Article
• Language: English
• Published: United States 01.11.2017
• Subjects: Animals; Antibodies - metabolism; Antigens - immunology; Antigens - metabolism; Datasets as Topic; Flow Cytometry - instrumentation; Flow Cytometry - methods; Fluorescence; Fluorescent Dyes - chemistry; Humans; Lasers - statistics & numerical data; Single-Cell Analysis; high dimensional; flow cytometry; spreading error; panel design; fluorescence; polychromatic
• ISSN: 1934-368X
• DOI: 10.1002/cpim.37

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.