CyTOF® for the Masses

Abstract

Mass cytometry has revolutionized immunophenotyping, particularly in exploratory settings where simultaneous breadth and depth of characterization of immune populations is needed with limited samples such as in preclinical and clinical tumor immunotherapy. Mass cytometry is also a powerful tool for single-cell immunological assays, especially for complex and simultaneous characterization of diverse intratumoral immune subsets or immunotherapeutic cell populations. Through the elimination of spectral overlap seen in optical flow cytometry by replacement of fluorescent labels with metal isotopes, mass cytometry allows, on average, robust analysis of 60 individual parameters simultaneously. This is, however, associated with significantly increased complexity in the design, execution, and interpretation of mass cytometry experiments. To address the key pitfalls associated with the fragmentation, complexity, and analysis of data in mass cytometry for immunologists who are novices to these techniques, we have developed a comprehensive resource guide. Included in this review are experiment and panel design, antibody conjugations, sample staining, sample acquisition, and data pre-processing and analysis. Where feasible multiple resources for the same process are compared, allowing researchers experienced in flow cytometry but with minimal mass cytometry expertise to develop a data-driven and streamlined project workflow. It is our hope that this manuscript will prove a useful resource for both beginning and advanced users of mass cytometry.

Cytokine and chemokine map of peripheral specific immune cell subsets in Parkinson's disease

“…Second, although CyTOF data can be highly sensitive, more data are required to limit the effect of false positives 32 . Third, future studies should isolate immune cells of interest within each subpopulation to verify their function and potential mechanism in PD.…”

Dormant cancer cells: programmed quiescence, senescence, or both?

“…The aforementioned studies largely leverage the rapidly advancing '-omics' technologies, which have been, and will continue to be, critical in providing novel ways to answer questions that were previously unanswerable. The advent of single-cell RNA sequencing (scRNAseq), cytometry by time of flight (CyTOF), matrix-assisted laser desorption/ionization-imaging mass spectrometry (MALDI-IMS), digital special profiling (DSP), and other techniques now make it possible to resolve biological systems spatially (in the case of MALDI-IMS and DSP), and at the single-cell level (with scRNAseq, CyTOF) [122][123][124][125]. Coupled to novel molecular technologies such as techniques that enable barcoding and lineage tracing of individual cells, it is also possible to track these biological processes spatiotemporally (reviewed Fig.…”

Emerging insights and challenges for understanding T cell function through the proteome

“…The introduction of spectral flow cytometry has expanded upon the limitations of traditional, polychromatic flow cytometry to enable visualization of up to 40 parameters on immune cells ( 8 ). Cytometry time of flight (CyTOF) is a variation of flow cytometry in which antibodies are labeled with heavy metal ion tags in lieu of fluorophores ( 9 ). CyTOF is a powerful tool to quantify labeled targets with simultaneous visualization of up to 60 individual parameters at once and is particularly useful for in-depth analysis of immune populations with limited samples.…”