Acquisition of High-Quality Spectral Flow Cytometry Data.

ABSTRACT

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.