A deep profiler's guide to cytometry

doi:10.1016/j.it.2012.02.010

Trends in Immunology

Volume 33, Issue 7, July 2012, Pages 323-332

https://doi.org/10.1016/j.it.2012.02.010 Get rights and content

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.

Section snippets

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,

Technical achievements that led to PFC

The development of PFC required multiple stepwise advancements in hardware and reagents. For example, the earliest fluorescence-based cytometers used arc lamps, developed originally for microscopy, emitting light at a broad spectrum of wavelengths [1]. This light interfered with fluorochrome-derived signals, therefore, arc-lamps were not easily used for multi-color detection. By 1974, in the Herzenberg Laboratory at Stanford University, argon lasers, emitting a single wavelength (488 nm) were

The post-fluorescence era: mass cytometry

A new platform has been developed that couples flow cytometry with mass spectrometry. This technology, known as mass cytometry, offers single-cell analysis of at least 45 simultaneous parameters without fluorescent agents or interference from spectral overlap (Figure 2). For this, stable (nonradioactive) isotopes of nonbiological, rare earth metals are used as reporters. By exploiting the resolution, sensitivity and dynamic range of mass spectrometry on a time-scale that allows the measurement

Analysis of multiplexed, multiparametric data

Rapid increases in the numbers of measurable single-cell parameters, both in flow and mass cytometry, have brought a daunting increase in the complexity of the data. Analysis of flow cytometry data is typically manual, performed in one or two dimensions at a time by selecting subsets of interest from parent populations. This approach is not scalable, and suffers from individual user bias (Figure 5a). Moreover, it requires prior knowledge of the cell type of interest, so unexpected cell types

Concluding remarks

Over the past 40 years, continual improvement in single-cell analysis technologies has driven our investigation and understanding of immunology and stem cell biology. Pushing the multiparameter limits of fluorescence-based analysis has led to unprecedented studies of regulatory signaling in both the healthy and diseased hematopoietic system. It has also identified many distinct immune cell subsets – most of which have no assigned function. Now, next-generation, mass cytometry instrumentation

Conflict of interest

M.R. receives royalties on the sale of FlowJo software and Cy7APC fluorescent reagents. G.P.N. owns stock and is a paid consultant with DVS Sciences (CyTOF manufacturer) and is a paid consultant with Becton Dickinson, a purveyor of reagents central to both cytometry platforms.

Acknowledgments

M.R. and P.K.C. are supported by the Intramural Research Program of the NIAID, NIH, and by the Collaboration for AIDS Vaccine Discovery (CAVD), Grant #OPP1032325, from the Bill and Melinda Gates Foundation. S.C.B. is supported by the Damon Runyon Cancer Research Foundation Fellowship (DRG-2017-09). G.P.N. is supported by the Rachford and Carlota A. Harris Endowed Professorship and grants from U19 AI057229, P01 CA034233, HHSN272200700038C, 1R01CA130826, CIRM DR1-01477 and RB2-01592, NCI RFA CA

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Beneficial from the strengths in sensitivity, elemental selectivity and multiplex capability, inductively coupled plasma mass spectrometry (ICP-MS) has emerged as a powerful tool for determination of disease biomarkers in clinical practice. In this field, specific biomolecular interactions between analytes and metal tags greatly facilitate precise target recognition and probe binding in complex biomatrices, and this enables the accuracy of ICP-MS bioassay for diagnostic applications. This review aims to underscore the effectiveness of specific bio-interactions in advancing element-tagged ICP-MS technique for biomarker quantification, cell analysis and tissue imaging. Recent progresses of elemental ICP-MS bioassay in clinical practice will be reviewed in the aspects of elemental-tagging approach, specific bio-interaction type and strength, time-resolved ICP-MS, laser ablation ICP-MS, as well as its prospects and future challenges.
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Mass cytometry permits the high dimensional analysis of complex biological samples; however, some techniques are not yet integrated into the mass cytometry workflow due to reagent availability. The use of self-labeling protein systems, such as HaloTag, are one such application. Here, we describe the design and implementation of the first mass cytometry ligands for use with HaloTag. “Click”-amenable HaloTag warheads were first conjugated onto poly(l-lysine) or poly(acrylic acid) polymers that were then functionalized with diethylenetriaminepentaacetic acid (DTPA) lutetium metal chelates. Kinetic analysis of the HaloTag labeling rates demonstrated that the structure appended to the 1-chlorohexyl warhead was key to success. A construct with a diethylene glycol spacer appended to a benzamide gave similar rates (k _obs ∼ 10² M^–1 s^–1), regardless of the nature of the polymer. Comparison of the polymer with a small molecule chelate having rapid HaloTag labeling kinetics (k _obs ∼ 10⁴ M^–1 s^–1) suggests the polymers significantly reduced the HaloTag labeling rate. HEK293T cells expressing surface-exposed GFP-HaloTag fusions were labeled with the polymeric constructs and ¹⁷⁵Lu content measured by cytometry by time-of-flight (CyTOF). Robust labeling was observed; however, significant nonspecific binding of the constructs to cells was also present. Heavily pegylated polymers demonstrated that nonspecific binding could be reduced to allow cells bearing the HaloTag protein to be distinguished from nonexpressing cells.
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This research reveals the capability of ICP-OES in single-cell analysis for micro-sized cells which was made possible by the new conical ICP torch and the new sample introduction system. The ability to detect calcium in single mammalian cells enables the first ever application of this technique to assess the impact of the Yoda1 activator on the calcium level in osteocytes.
Advances in Clinical Mass Cytometry
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Single-cell technologies have revealed the extensive heterogeneity and complexity of the immune system. Systems biology approaches in immunology have taken advantage of the high-parameter, high-throughput data and analyzed immune cell types in a ‘bottom-up’ data-driven method. This approach has discovered previously unrecognized cell types and functions. Especially for human immunology, in which experimental manipulations are challenging, systems approach has become a successful means to investigate physiologically relevant contexts. This review focuses on the recent findings in lymphocyte biology, from their development, differentiation into subsets, and heterogeneity in their functions, enabled by these systems approaches. Furthermore, we review examples of the application of findings from systems approach studies and discuss how now to leave the rich dataset in the curse of high dimensionality.

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Published by Elsevier Ltd.

Trends in Immunology

ReviewA deep profiler's guide to cytometry

Section snippets

The case for deep profiling

Technical achievements that led to PFC

The post-fluorescence era: mass cytometry

Analysis of multiplexed, multiparametric data

Concluding remarks

Conflict of interest

Acknowledgments

Blood

J. Immunol. Methods

Blood

Blood

Spectrochim. Acta Part B: Atomic Spectrosc.

J. Immunol. Methods

J. Immunol. Methods

Immunity

Methods Cell Biol.

The Fluorescent Cell Photometer: A New Method for the Rapid Measurement of Biological Cells Stained with Fluorescent Dyes

Development and application of a rapid cell sorter

Clin. Chem.

Future directions for flow cytometry

J. Histochem. Cytochem.

Dual-laser flow cytometry of single mammalian cells

J. Histochem. Cytochem.

Immunofluorescence measurement in a flow cytometer using low-power helium-neon laser excitation

Cytometry

Fluorescent phycobiliprotein conjugates for analyses of cells and molecules

J. Cell Biol.

Lasers in flow cytometry

Methods Cell Biol.

Single laser three color immunofluorescence staining procedures based on energy transfer between phycoerythrin and cyanine 5

Cytometry

Cy7PE and Cy7APC: bright new probes for immunofluorescence

Cytometry

Quantitative comparison of long-wavelength Alexa Fluor dyes to Cy dyes: fluorescence of the dyes and their bioconjugates

J. Histochem. Cytochem.

8 color, 10-parameter flow cytometry to elucidate complex leukocyte heterogeneity

Cytometry

11-color, 13-parameter flow cytometry: identification of human naive T cells by phenotype, function, and T-cell receptor diversity

Nat. Med.

Quantum dot semiconductor nanocrystals for immunophenotyping by polychromatic flow cytometry

Nat. Med.

Chemistry Nobel, getting a charge out of plastics

Science

Cell sorting: automated separation of mammalian cells as a function of intracellular fluorescence

Science

Differential fluorochromasia of human lymphocytes as measured by flow cytometry

J. Histochem. Cytochem.

Continuous cultures of fused cells secreting antibody of predefined specificity

Nature

Multiparameter flow cytometry in the diagnosis and management of acute leukemia

Arch. Pathol. Lab. Med.

The regulation and role of T follicular helper cells in immunity

Immunology

Mechanisms controlling Th17 cytokine expression and host defense

J. Leukoc. Biol.

A human memory T cell subset with stem cell-like properties

Nat. Med.

Review
A deep profiler's guide to cytometry