Multiparameter Phenotyping of Human PBMCs Using Mass Cytometry

  • Michael D. Leipold
  • Evan W. Newell
  • Holden T. Maecker
Part of the Methods in Molecular Biology book series (MIMB, volume 1343)


The standard for single-cell analysis of phenotype and function in recent decades has been fluorescence flow cytometry. Mass cytometry is a newer technology that uses heavy metal ions, rather than fluorochromes, as labels for probes such as antibodies. The binding of these ion-labeled probes to cells is quantitated by mass spectrometry. This greatly increases the number of phenotypic and functional markers that can be probed simultaneously. Here, we review topics that must be considered when adapting existing flow cytometry panels to mass cytometry analysis. We present a protocol and representative panels for surface phenotyping and intracellular cytokine staining (ICS) assays.

Key words

Mass cytometry CyTOF Immunophenotyping Panel design 



The authors thank Sean Bendall for helpful discussions, and Sheena Gupta and Meena Malipatlolla for contributing example data. Development of this protocol was funded in part by grant 2 U19 AI057229 S4 from the National Institutes of Health.


  1. 1.
    Kalisky T, Quake SR (2011) Single-cell genomics. Nat Methods 8:311–314PubMedCrossRefGoogle Scholar
  2. 2.
    Ornatsky O, Bandura D, Baranov V, Nitz M, Winnik MA, Tanner S (2010) Highly multiparametric analysis by mass cytometry. J Immunol Methods 36:1–20CrossRefGoogle Scholar
  3. 3.
    Bandura DR, Baranov VI, Ornatsky OI, Antonov A, Kinach R, Lou X, Pavlov S, Vorobiev S, Dick JE, Tanner SD (2009) Mass cytometry: technique for real time single cell multitarget immunoassay based on inductively coupled plasma time-of-flight mass spectrometry. Anal Chem 81:6813–6822PubMedCrossRefGoogle Scholar
  4. 4.
    Newell EW, Sigal N, Bendall SC, Nolan GP, Davis MM (2012) Cytometry by time-of-flight shows combinatorial cytokine expression and virus-specific cell niches within a continuum of CD8(+) T cell phenotypes. Immunity 36:142–152PubMedPubMedCentralCrossRefGoogle Scholar
  5. 5.
    Bendall SC, Simonds EF, Qiu P, Amir el AD, Krutzik PO, Finck R, Bruggner RV, Melamed R, Trejo A, Ornatsky OI, Balderas RS, Plevritis SK, Sachs K, Pe'er D, Tanner SD, Nolan GP (2011) Single-cell mass cytometry of differential immune and drug responses across a human hematopoietic continuum. Science 332:687–696PubMedPubMedCentralCrossRefGoogle Scholar
  6. 6.
    Bodenmiller B, Zunder ER, Finck R, Chen TJ, Savig ES, Bruggner RV, Simonds EF, Bendall SC, Sachs K, Krutzik PO, Nolan GP (2012) Multiplexed mass cytometry profiling of cellular states perturbed by small-molecule regulators. Nat Biotechnol 30:858–867PubMedPubMedCentralCrossRefGoogle Scholar
  7. 7.
    Behbehani GK, Bendall SC, Clutter MR, Fantl WJ, Nolan GP (2012) Single-cell mass cytometry adapted to measurements of the cell cycle. Cytometry A 81:552–566PubMedPubMedCentralCrossRefGoogle Scholar
  8. 8.
    Fienberg HG, Simonds EF, Fantl WJ, Nolan GP, Bodenmiller B (2012) A platinum-based covalent viability reagent for single-cell mass cytometry. Cytometry A 81:467–475PubMedCrossRefGoogle Scholar
  9. 9.
    Maecker HT (2009) Multiparameter flow cytometry monitoring of T cell responses. Methods Mol Biol 485:375–391PubMedPubMedCentralCrossRefGoogle Scholar
  10. 10.
    Maecker HT, Frey T, Nomura LE, Trotter J (2004) Selecting fluorochrome conjugates for maximum sensitivity. Cytometry A 62:169–173PubMedCrossRefGoogle Scholar
  11. 11.
    Rundberg Nilsson A, Bryder D, Pronk CJ (2013) Frequency determination of rare populations by flow cytometry: a hematopoietic stem cell perspective. Cytometry A 83:721–727PubMedCrossRefGoogle Scholar
  12. 12.
    Ornatsky OI, Kinach R, Bandura DR, Lou X, Tanner SD, Baranov VI, Nitz M, Winnik MA (2008) Development of analytical methods for multiplex bio-assay with inductively coupled plasma mass spectrometry. J Anal At Spectrom 23:463–469PubMedPubMedCentralCrossRefGoogle Scholar
  13. 13.
    Lovelace P, Maecker HT (2010) Multiparameter intracellular cytokine staining. Methods Mol Biol 699:165–178CrossRefGoogle Scholar
  14. 14.
    Leipold MD, Maecker HT (2012) Mass cytometry: protocol for daily tuning and running cell samples on a CyTOF mass cytometer. J Vis Exp. doi: 10.3791/4398 PubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Michael D. Leipold
    • 1
  • Evan W. Newell
    • 2
  • Holden T. Maecker
    • 3
  1. 1.Institute for Immunity, Transplantation, and InfectionStanford UniversityStanfordUSA
  2. 2.Singapore Immunology NetworkSingaporeSingapore
  3. 3.Institute for Immunity, Transplantation, and Infection, Department of Microbiology and ImmunologyStanford UniversityStanfordUSA

Personalised recommendations