Discrimination of Seven Immune Cell Subsets by Two-fluorochrome Flow Cytometry

Maria Letizia Giardino Torchia; Raffaello Cimbro

doi:10.3791/58955

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Method Article

Discrimination of Seven Immune Cell Subsets by Two-fluorochrome Flow Cytometry

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DOI:

10.3791/58955

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March 5th, 2019

Maria Letizia Giardino Torchia¹ , Raffaello Cimbro²

¹Oncology Research, Medimmune, LLC, ²Division of Rheumatology, Johns Hopkins University School of Medicine

Summary

Here, we present a flow cytometric protocol to identify CD4⁺ and CD8⁺ T cells, γδ T cells, B cells, NK cells and monocytes in human peripheral blood by using only two fluorochromes instead of seven. With this approach, five additional markers can be recorded on most flow cytometers.

Abstract

Immune cell characterization heavily relies on multicolor flow cytometry to identify subpopulations based on differential expression of surface markers. Setup of a classic multicolor panel requires high-end instruments, custom labeled antibodies, and careful study design to minimize spectral overlap. We developed a multiparametric analysis to identify major human immune populations (CD4⁺ and CD8⁺ T cells, γδ T cells, B cells, NK cells and monocytes) in peripheral blood by combining seven lineage markers using only two fluorochromes. Our strategy is based on the observation that lineage markers are constantly expressed in a unique combination by each cell population. Combining this information with a careful titration of the antibodies allows investigators to record five additional markers, expanding the optical limit of most flow cytometers. Head-to-head comparison demonstrated that the vast majority of immune cell populations in peripheral blood can be characterized with comparable accuracy between our method and the classic "one fluorochrome-one marker approach", although the latter is still more precise for identifying populations such as NKT cells and γδ T cells. Combining seven markers using two fluorochromes allows for the analysis of complex immune cell populations and clinical samples on affordable 6-10 fluorochrome flow cytometers, and even on 2-3 fluorochrome field instruments in areas with limited resources. High-end instruments can also benefit from this approach by using extra fluorochromes to accomplish deeper flow cytometry analysis. This approach is also very well suited for screening several cell populations in the case of clinical samples with limited number of cells.

Introduction

Flow cytometry is a technique that was developed to analyze multiple parameters on single particles at a rate of several thousand of events per second¹. Examples of specimens analyzed by flow cytometry include, but are not limited to, cells, beads, bacteria, vesicles and chromosomes. A fluidic system directs particles at the interrogation point where each particle intersects its path with one or more lasers, and multiple parameters are recorded for further analysis. Forward and side scatters, generated by scattering of the pure laser light, are used to identify the target population and retrieve information about the relative size and internal ....