High‐Dimensional Fluorescence Cytometry

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.

Panel Design and Optimization for High‐Dimensional Immunophenotyping Assays Using Spectral Flow Cytometry

“…As a general rule, Cytek Biosciences recommends acquisition of 5000 events for beads and 1000 events for cells; however, it is important to have at least 500 events each in the positive and negative gates. Certain conditions, such as low antigen density or rare positive events, might greatly benefit from use of compensation beads (see Current Protocols article; Ashhurst et al., 2017). Either compensation beads or cells can be used as single‐stained controls in the same experiment as long as the correct internal negative control is used (e.g., unstained compensation beads or cells with similar AF, respectively).…”

“…SRCs should also be treated the same way as fully stained samples, as fixation steps or pH variations can alter the spectral profiles of some fluorophores (see Current Protocols article; Ashhurst et al., 2017). Such treatments can also affect the fluorescent signature of compensation beads.…”

“…Determining SE is crucial when building high-dimensional fluorescence panels. Factors that contribute to the amount of SE from a given fluorophore into another fluorescence detector have been well described (see Current Protocols article; Ashhurst et al, 2017;Nguyen, Perfetto, Mahnke, Chattopadhyay, & Roederer, 2013). It is important to note that the CSI matrix is highly specific to the instrument setup and the fluorophores used and needs to be modified if additional or alternative fluorophores or another instrument is used.…”

“…This protocol was built around the three-laser Aurora but can easily be adapted to any spectral flow cytometer with other specifications. Although the design of spectral flow cytometry panels follows similar steps to those described for conventional flow cytometry (see Current Protocols article; Ashhurst, Smith, & King, 2017;Brummelman et al, 2019), important differences and additional considerations apply for spectral flow cytometry, as described in this protocol (for an overview, see Fig. 1).…”

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

“…In this section, we provide a simple step-by-step protocol for the successful design of a high-dimensional spectral flow cytometry panel. While the design of spectral flow cytometry panels follows similar steps to those described for conventional flow cytometry 5,15 , important differences and additional considerations apply for spectral flow cytometry, which are described in this protocol.…”