Acquisition of High-Quality Spectral Flow Cytometry Data

Curr Protoc Cytom. 2020 Jun;93(1):e74. doi: 10.1002/cpcy.74.


Flow cytometry allows the visualization of physical, functional, and/or biological properties of cells including antigens, cytokines, size, and complexity. With increasingly large flow cytometry panels able to analyze up to 50 parameters, there is a need to standardize flow cytometry protocols to achieve high-quality data that can be input into analysis algorithms. Without this clean data, algorithms may incorrectly categorize the cell populations present in the samples. In this protocol, we outline a comprehensive methodology to prepare samples for polychromatic flow cytometry. The use of multiple washing steps and rigorous controls creates high-quality data with good separation between cell populations. Experimental data acquired using this protocol can be analyzed via computational algorithms that perform end-to-end analysis. © 2020 by John Wiley & Sons, Inc. Basic Protocol 1: Preparation of single-cell suspension for flow cytometry Support Protocol 1: Lung preparation Support Protocol 2: Counting cells on a flow cytometer Basic Protocol 2: Surface and intracellular flow cytometry staining Support Protocol 3: Single-color bead controls.

Keywords: controls; flow cytometry; high-dimensional flow cytometry data; high-quality data; immunology; phenotyping; sample preparation.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Animals
  • Cell Count
  • Flow Cytometry / methods*
  • Flow Cytometry / standards*
  • Intracellular Space / metabolism
  • Lung / cytology
  • Mice, Inbred C57BL
  • Single-Cell Analysis
  • Spleen / cytology
  • T-Lymphocytes / immunology