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A deep profiler’s guide to cytometry
Sean C. Bendall1, Garry P. Nolan1, Mario Roederer2 and Pratip K. Chattopadhyay2
1 Baxter Laboratory in Stem Cell Biology, Department of Microbiology and Immunology, Stanford University, Stanford, CA 94305,
USA
2 ImmunoTechnology Section, Vaccine Research Center, NIAID, NIH, Bethesda, MD, USA
In recent years, advances in technology have provided us
with tools to quantify the expression of multiple genes in
individual cells. The ability to measure simultaneously
multiple genes in the same cell is necessary to resolve
the great diversity of cell subsets, as well as to define
their function in the host. Fluorescence-based flow cytometry
is the benchmark for this; with it, we can quantify
18 proteins per cell, at >10 000 cells/s. Mass cytometry is
a new technology that promises to extend these capabilities
significantly. Immunophenotyping by mass spectrometry
provides the ability to measure >36 proteins at
a rate of 1000 cells/s. We review these cytometric technologies,
capable of high-content, high-throughput single-
cell assays.
The case for deep profiling
To understand the biological actions of cells and their mechanisms
of differentiation, we must understand how phenotype
and function are structured across diverse cell types
and tissues. This structure can be perturbed by innate or
infectious sources, which may drive disease pathogenesis;
therefore, understanding it is crucial for identifying treatments
and preventions. Great cellular diversity underlies
this organization, so measurements taken at the single-cell
level that encompass RNA, protein and glycan species (‘high
content’) across many cells (‘high throughput’) will greatly
aid our formulation of a more comprehensive understanding.
In many respects, this is walking the path previously
trodden by genomics and proteomics – long accustomed to
thinking about many target markers per experiment. However,
traditional single-cell analysis has focused on many
cells and a few parameters per experiment. As we delve into
more complex cellular systems, such as cellular signaling
networks or T cell functional responses, we must reorient
this thinking to consider many parameters in many cells; in
essence, ‘deep profiling’ every single cell from a representative
population of cells.
Among well-established technologies for cellular analysis,
flow cytometry is unique for its ability to interrogate
rapidly multiple biologic signatures (protein epitopes,
nucleic acids, ion concentrations) simultaneously within a
single cell. Over the past 40 years, since the introduction of
the first fluorescence-based flow cytometers, the maximum
number of proteins that can be simultaneously measured
has progressively increased. These advances can be attributed
to parallel