Immunophenotyping by Mass Cytometry

  • Gregory K. BehbehaniEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 2032)


Mass cytometry is a novel technology similar to flow cytometry in which antibodies are tagged with heavy metal molecules rather than fluorophores and then detected with time-of-flight mass spectrometry. This enables measurement of up to 50 simultaneous parameters with no autofluorescent background and little or no spillover or required compensation. Mass cytometry has tremendous potential for the analysis of highly complex research or clinical samples and can measure 40–50 immunophenotypic markers at a time. This chapter describes most of the commonly used methods for performing basic immunophenotyping experiments by mass cytometry, and how this can be combined with measurement of cellular functional properties.

Key words

Immunophenotyping Mass cytometry CyTOF Flow cytometry Minimal residual disease Acute leukemia Aberrant marker expression 


  1. 1.
    Bendall SC, Nolan GP, Roederer M, Chattopadhyay PK (2012) A deep profiler’s guide to cytometry. Trends Immunol 33(7):323–332CrossRefGoogle Scholar
  2. 2.
    Tanner SD, Baranov VI, Ornatsky OI, Bandura DR, George TC (2013) An introduction to mass cytometry: fundamentals and applications. Cancer Immunol Immunother 62(5):955–965CrossRefGoogle Scholar
  3. 3.
    Spitzer MH, Nolan GP (2016) Mass cytometry: single cells, many features. Cell 165(4):780–791CrossRefGoogle Scholar
  4. 4.
    Bjornson ZB, Nolan GP, Fantl WJ (2013) Single-cell mass cytometry for analysis of immune system functional states. Curr Opin Immunol 25(4):484–494CrossRefGoogle Scholar
  5. 5.
    Di Palma S, Bodenmiller B (2015) Unraveling cell populations in tumors by single-cell mass cytometry. Curr Opin Biotechnol 31:122–129CrossRefGoogle Scholar
  6. 6.
    Behbehani GK (2017) Applications of mass cytometry in clinical medicine: the promise and perils of clinical CyTOF. Clin Lab Med 37(4):945–964CrossRefGoogle Scholar
  7. 7.
    Rahman AH, Tordesillas L, Berin MC (2016) Heparin reduces nonspecific eosinophil staining artifacts in mass cytometry experiments. Cytometry A 89(6):601–607CrossRefGoogle Scholar
  8. 8.
    Frei AP, Bava F-A, Zunder ER et al (2016) Highly multiplexed simultaneous detection of RNAs and proteins in single cells. Nat Methods 13:269CrossRefGoogle Scholar
  9. 9.
    Behbehani GK, Bendall SC, Clutter MR, Fantl WJ, Nolan GP (2012) Single-cell mass cytometry adapted to measurements of the cell cycle. Cytometry A 81(7):552–566CrossRefGoogle Scholar
  10. 10.
    Edgar LJ, Vellanki RN, Halupa A, Hedley D, Wouters BG, Nitz M (2014) Identification of hypoxic cells using an organotellurium tag compatible with mass cytometry. Angew Chem Int Ed Engl 53(43):11473–11477CrossRefGoogle Scholar
  11. 11.
    Han G, Spitzer MH, Bendall SC, Fantl WJ, Nolan GP (2018) Metal-isotope-tagged monoclonal antibodies for high-dimensional mass cytometry. Nat Protoc 13(10):2121–2148CrossRefGoogle Scholar
  12. 12.
    Chang Q, Ornatsky OI, Koch CJ et al (2015) Single-cell measurement of the uptake, intratumoral distribution and cell cycle effects of cisplatin using mass cytometry. Int J Cancer 136(5):1202–1209CrossRefGoogle Scholar
  13. 13.
    Chevrier S, Crowell HL, Zanotelli VRT, Engler S, Robinson MD, Bodenmiller B (2018) Compensation of signal spillover in suspension and imaging mass cytometry. Cell Syst 6(5):612–620 e615CrossRefGoogle Scholar
  14. 14.
    Fienberg HG, Simonds EF, Fantl WJ, Nolan GP, Bodenmiller B (2012) A platinum-based covalent viability reagent for single-cell mass cytometry. Cytometry A 81(6):467–475CrossRefGoogle Scholar
  15. 15.
    Zunder ER, Finck R, Behbehani GK et al (2015) Palladium-based mass tag cell barcoding with a doublet-filtering scheme and single-cell deconvolution algorithm. Nat Protocols 10(2):316–333CrossRefGoogle Scholar
  16. 16.
    Ornatsky OI, Lou X, Nitz M et al (2008) Study of cell antigens and intracellular DNA by identification of element-containing labels and metallointercalators using inductively coupled plasma mass spectrometry. Anal Chem 80(7):2539–2547CrossRefGoogle Scholar
  17. 17.
    Bendall SC, Simonds EF, Qiu P et al (2011) Single-cell mass cytometry of differential immune and drug responses across a human hematopoietic continuum. Science 332(6030):687–696CrossRefGoogle Scholar
  18. 18.
    Behbehani GK (2018) Cell cycle analysis by mass cytometry. In: Lacorazza HD (ed) Cellular quiescence: methods and protocols. Springer New York, New York, NY, pp 105–124CrossRefGoogle Scholar
  19. 19.
    Behbehani GK, Thom C, Zunder ER et al (2014) Transient partial permeabilization with saponin enables cellular barcoding prior to surface marker staining. Cytometry A 85(12):1011–1019CrossRefGoogle Scholar
  20. 20.
    Hartmann FJ, Simonds EF, Bendall SC (2018) A universal live cell barcoding-platform for multiplexed human single cell analysis. Sci Rep 8(1):10770CrossRefGoogle Scholar
  21. 21.
    Chow S, Hedley D, Grom P, Magari R, Jacobberger JW, Shankey TV (2005) Whole blood fixation and permeabilization protocol with red blood cell lysis for flow cytometry of intracellular phosphorylated epitopes in leukocyte subpopulations. Cytometry A 67(1):4–17CrossRefGoogle Scholar
  22. 22.
    Finck R, Simonds EF, Jager A et al (2013) Normalization of mass cytometry data with bead standards. Cytometry A 83(5):483–494CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of Internal Medicine, Division of Hematology, James Cancer HospitalThe Ohio State UniversityColumbusUSA

Personalised recommendations