Skip to main content
SpringerLink
Log in
Book cover

Mass Cytometry pp 93–108Cite as

Surface Barcoding of Live PBMC for Multiplexed Mass Cytometry

  • Protocol
  • First Online:

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1989))

Abstract

Sample barcoding is a powerful method for harmonizing mass cytometry data. By assigning a unique combination of barcode labels to each cell sample, a set of individual samples can be pooled and further processed and acquired as a large, single sample. For assays that require uncompromised profiling of cell-surface markers on live cells, barcoding by metal-labeled antibodies targeting cell-surface epitopes is the barcoding approach of choice. Here we provide an optimized and validated protocol for cell-surface barcoding of ten PBMC samples with palladium-labeled β2-microglobulin (B2M) antibodies used in a 5-choose-2 barcoding scheme, for subsequent immune phenotyping by mass cytometry. We further provide details on the generation of palladium-labeled antibodies utilizing amine-reactive isothiocyanobenzyl-EDTA (ITCB-EDTA) that permits the implementation of antibody-based barcoding not interfering with lanthanide channels typically used for analyte detection in mass cytometry assays.

This is a preview of subscription content, log in via an institution.

Protocol
USD   49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD   189.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Hardcover Book
USD   249.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Learn about institutional subscriptions

Springer Nature is developing a new tool to find and evaluate Protocols. Learn more

References

  1. 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):687–696. https://doi.org/10.1126/science.1198704

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Levine JH, Simonds EF, Bendall SC et al (2015) Data-driven phenotypic dissection of AML reveals progenitor-like cells that correlate with prognosis. Cell 162(1):184–197. https://doi.org/10.1016/j.cell.2015.05.047

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Rao DA, Gurish MF, Marshall JL et al (2017) Pathologically expanded peripheral T helper cell subset drives B cells in rheumatoid arthritis. Nature 542(7639):110–114. https://doi.org/10.1038/nature20810

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. O’Gorman WE, Hsieh EW, Savig ES et al (2015) Single-cell systems-level analysis of human Toll-like receptor activation defines a chemokine signature in patients with systemic lupus erythematosus. J Allergy Clin Immunol 136(5):1326–1336. https://doi.org/10.1016/j.jaci.2015.04.008

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Gaudilliere B, Fragiadakis GK, Bruggner RV et al (2014) Clinical recovery from surgery correlates with single-cell immune signatures. Sci Transl Med 6(255):255ra131. https://doi.org/10.1126/scitranslmed.3009701

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Nair N, Mei HE, Chen SY et al (2015) Mass cytometry as a platform for the discovery of cellular biomarkers to guide effective rheumatic disease therapy. Arthritis Res Ther 17:127. https://doi.org/10.1186/s13075-015-0644-z

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Ermann J, Rao DA, Teslovich NC et al (2015) Immune cell profiling to guide therapeutic decisions in rheumatic diseases. Nat Rev Rheumatol 11(9):541–551. https://doi.org/10.1038/nrrheum.2015.71

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Krutzik PO, Nolan GP (2006) Fluorescent cell barcoding in flow cytometry allows high-throughput drug screening and signaling profiling. Nat Methods 3(5):361–368. https://doi.org/10.1038/nmeth872

    Article  CAS  PubMed  Google Scholar 

  9. Bodenmiller B, Zunder ER, Finck R et al (2012) Multiplexed mass cytometry profiling of cellular states perturbed by small-molecule regulators. Nat Biotechnol 30(9):858–867. https://doi.org/10.1038/nbt.2317

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Zivanovic N, Jacobs A, Bodenmiller B (2014) A practical guide to multiplexed mass cytometry. Curr Top Microbiol Immunol 377:95–109. https://doi.org/10.1007/82_2013_335

    Article  CAS  PubMed  Google Scholar 

  11. 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 Protoc 10(2):316–333. https://doi.org/10.1038/nprot.2015.020

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. McCarthy RL, Mak DH, Burks JK et al (2017) Rapid monoisotopic cisplatin based barcoding for multiplexed mass cytometry. Sci Rep 7(1):3779. https://doi.org/10.1038/s41598-017-03610-2

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Catena R, Ozcan A, Zivanovic N et al (2016) Enhanced multiplexing in mass cytometry using osmium and ruthenium tetroxide species. Cytometry A 89(5):491–497. https://doi.org/10.1002/cyto.a.22848

    Article  CAS  PubMed  Google Scholar 

  14. 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):1011–1019. https://doi.org/10.1002/cyto.a.22573

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Dzangue-Tchoupou G, Corneau A, Blanc C et al (2018) Analysis of cell surface and intranuclear markers on non-stimulated human PBMC using mass cytometry. PLoS One 13(3):e0194593. https://doi.org/10.1371/journal.pone.0194593

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Gaudilliere B, Ganio EA, Tingle M et al (2015) Implementing mass cytometry at the bedside to study the immunological basis of human diseases: distinctive immune features in patients with a history of term or preterm birth. Cytometry A 87(9):817–829. https://doi.org/10.1002/cyto.a.22720

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Mei HE, Leipold MD, Schulz AR et al (2015) Barcoding of live human peripheral blood mononuclear cells for multiplexed mass cytometry. J Immunol 194(4):2022–2031. https://doi.org/10.4049/jimmunol.1402661

    Article  CAS  PubMed  Google Scholar 

  18. Mei HE, Leipold MD, Maecker HT (2016) Platinum-conjugated antibodies for application in mass cytometry. Cytometry A 89(3):292–300. https://doi.org/10.1002/cyto.a.22778

    Article  CAS  PubMed  Google Scholar 

  19. Lavin Y, Kobayashi S, Leader A et al (2017) Innate immune landscape in early lung adenocarcinoma by paired single-cell analyses. Cell 169(4):750–765.e717. https://doi.org/10.1016/j.cell.2017.04.014

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Mrdjen D, Pavlovic A, Hartmann FJ et al (2018) High-dimensional single-cell mapping of central nervous system immune cells reveals distinct myeloid subsets in health, aging, and disease. Immunity 48(2):380–395.e386. https://doi.org/10.1016/j.immuni.2018.01.011

    Article  CAS  PubMed  Google Scholar 

  21. Lai L, Ong R, Li J et al (2015) A CD45-based barcoding approach to multiplex mass-cytometry (CyTOF). Cytometry A 87(4):369–374. https://doi.org/10.1002/cyto.a.22640

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Hartmann FJ, Bernard-Valnet R, Queriault C et al (2016) High-dimensional single-cell analysis reveals the immune signature of narcolepsy. J Exp Med 213(12):2621–2633. https://doi.org/10.1084/jem.20160897

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Lawson DA, Bhakta NR, Kessenbrock K et al (2015) Single-cell analysis reveals a stem-cell program in human metastatic breast cancer cells. Nature 526(7571):131–135. https://doi.org/10.1038/nature15260

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Hartmann FJ, Simonds EF, Bendall SC A Universal Live Cell Barcoding-Platform for Multiplexed Human Single Cell Analysis. PMID 30018331

    Google Scholar 

  25. Medina F, Segundo C, Campos-Caro A et al (2002) The heterogeneity shown by human plasma cells from tonsil, blood, and bone marrow reveals graded stages of increasing maturity, but local profiles of adhesion molecule expression. Blood 99(6):2154–2161

    Article  CAS  PubMed  Google Scholar 

  26. Cossarizza A, Chang HD, Radbruch A et al (2017) Guidelines for the use of flow cytometry and cell sorting in immunological studies. Eur J Immunol 47(10):1584–1797. https://doi.org/10.1002/eji.201646632

    Article  CAS  PubMed  Google Scholar 

  27. Finck R, Simonds EF, Jager A et al (2013) Normalization of mass cytometry data with bead standards. Cytometry A 83(5):483–494. https://doi.org/10.1002/cyto.a.22271

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Biesen R, Demir C, Barkhudarova F et al (2008) Sialic acid-binding Ig-like lectin 1 expression in inflammatory and resident monocytes is a potential biomarker for monitoring disease activity and success of therapy in systemic lupus erythematosus. Arthritis Rheum 58(4):1136–1145. https://doi.org/10.1002/art.23404

    Article  CAS  PubMed  Google Scholar 

  29. Rose T, Grutzkau A, Hirseland H et al (2013) IFNalpha and its response proteins, IP-10 and SIGLEC-1, are biomarkers of disease activity in systemic lupus erythematosus. Ann Rheum Dis 72(10):1639–1645. https://doi.org/10.1136/annrheumdis-2012-201586

    Article  CAS  PubMed  Google Scholar 

  30. Schulz AR, Stanislawiak S, Baumgart S et al (2017) Silver nanoparticles for the detection of cell surface antigens in mass cytometry. Cytometry A 91(1):25–33. https://doi.org/10.1002/cyto.a.22904

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

The authors would like to thank all members of the DRFZ mass cytometry group, Michael D. Leipold, PhD, for discussing antibody labeling strategies, Dr. Lars Fransecky for providing leukemia samples, and Dr. Petra Henklein for providing access to the lyophilizer.

Author information

Authors and Affiliations

  1. Mass Cytometry Lab, German Rheumatism Research Center (DRFZ), A Leibniz Institute, Berlin, Germany

    Axel Ronald Schulz & Henrik E. Mei

Authors
  1. Axel Ronald Schulz
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Henrik E. Mei
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Henrik E. Mei .

Editor information

Editors and Affiliations

  1. Discipline of Pathology, The University of Sydney, Camperdown, NSW, Australia

    Helen M. McGuire

  2. Sydney Cytometry Facility, The University of Sydney and Centenary Institute, Camperdown, NSW, Australia

    Thomas M. Ashhurst

Rights and permissions

Reprints and permissions

Copyright information

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

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Schulz, A.R., Mei, H.E. (2019). Surface Barcoding of Live PBMC for Multiplexed Mass Cytometry. In: McGuire, H., Ashhurst, T. (eds) Mass Cytometry. Methods in Molecular Biology, vol 1989. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9454-0_7

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-9454-0_7

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-4939-9453-3

  • Online ISBN: 978-1-4939-9454-0

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation