CyTOF: An Emerging Technology for Single‐Cell Proteomics in the Mouse

Abstract

The ability to analyze the proteome of single cells is critical for the advancement of studies of steady‐state and pathological processes. Mass cytometry, or CyTOF, combines principles of mass spectrometry and flow cytometry to enable single‐cell analysis of protein expression. CyTOF can simultaneously assess DNA content and proteins and has the capacity to measure 40 to 100 parameters in each cell. Applying this technology to tissues or cells on slides, termed imaging mass cytometry (IMC), allows for visualization of normal and diseased tissues in situ. The high‐dimensional proteomic analysis that can be undertaken with CyTOF and IMC has the potential to enhance our understanding of complex and heterogeneous developmental and disease pathways. This article will describe the CyTOF experimental workflow, including reagent selection, sample preparation, and data analysis. CyTOF is compared to flow cytometry, focusing on the strengths and weaknesses of these powerful techniques. Importantly, we review key studies in mouse models of human disease that highlight the strength of CyTOF and IMC to drive discovery research and therapeutic advancement. © 2021 Wiley Periodicals LLC.

Dynamic Interactions of Transcription Factors and Enhancer Reprogramming in Cancer Progression

“…This allows researchers to resolve changes in gene expression in different physical locations. This technology has been utilized to characterize unique interactions between tumor cells and the microenvironment, which would have otherwise been obscured by traditional RNA-seq (129). Other emerging approaches to study cell-cell interactions include, imaging-based mass spectrometry, CyTOF (cytometry by time-of-flight mass spectrometry) and imaging-coupled transcriptional profiling (130, 131).…”

Recent progress in cytometric technologies and their applications in ecotoxicology and environmental risk assessment

“…Since different metal ion tags have different atomic masses, they reach the detector at different time (hence the name CyTOF®, cytometry by time of flight). The CyTOF data are highly multi‐parametric and consist of distinctive and well separated metal tag abundance peaks that directly correlate with expression of labeled antigens [69, 70] (Figure 5C).…”

“…The MS can reportedly resolve up to 100 distinct channels that do not require spectral compensation, thus providing with unprecedented levels of multiplexing and high-content analysis[66,68]. The MS has been initially pioneered by University of Toronto and DVS Sciences and is currently available commercially as CyTOF ® (cytometry by time of flight; Fludigim Inc, San Francisco, CA, USA)[65,68,69].There are two variations of MS technology. First is a flow-based system where labeled cell sample in suspension is first nebulized into a fine aerosol[70] (Figure5C).…”

“…Since different metal ion tags have different atomic masses, they reach the detector at different time (hence the name CyTOF ® , cytometry by time of flight). The CyTOF data are highly multiparametric and consist of distinctive and well separated metal tag abundance peaks that directly correlate with expression of labeled antigens[69,70] (Figure5C).The imaging mass cytometry instead of aerosolized samples employs a laser beam that successively ablates small areas of samples (approximately 1 μm 2 ) such as adherent cell cultures, tissues biopsies, paraffin embedded sections, and tissue microarrays that are labeled F I G U R E 4 Software-based image cytometry enables to utilize cytometric data analysis strategies for images acquired using conventional fluorescence microscopes and automated imaging systems alike. (A) Ultra-high throughout cell cytotoxicity assay performed with a Live-Dead stain on ImageXpress Velos (Molecular Devices).…”

“…First is a flow-based system where labeled cell sample in suspension is first nebulized into a fine aerosol[70] (Figure5C). The nebulized sample passes then through an inductively coupled plasma (ICP) torch that creates an ion cloud[69][70][71] (Figure5C). The latter is injected into the time-of-flight mass spectroscopy (TOF-MS) detector.…”