CyTOF® for the Masses

Front Immunol. 2022 Apr 14;13:815828. doi: 10.3389/fimmu.2022.815828. eCollection 2022.


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.

Keywords: CyTOF; barcoding; data analysis; experimental design; mass cytometry; panel design; protocol; workflow.

Publication types

  • Review

MeSH terms

  • Antibodies*
  • Flow Cytometry / methods
  • Immunophenotyping
  • Single-Cell Analysis* / methods
  • Staining and Labeling


  • Antibodies