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Method for Tagging Antibodies with Metals for Mass Cytometry Experiments

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Mass Cytometry

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

Abstract

Mass cytometers are time-of-flight (TOF) mass spectrometer-coupled flow cytometers (known as CyTOFs) that quantify the abundance of metal-tagged antibodies (Abs) or other cellular probes within single cell suspensions or laser-ablated tissue sections. While many strategies exist for covalently crosslinking to proteins, the Fluidigm MaxPar® process is currently the most widely used and involves first loading a metal-chelating polymer with an elementally and isotopically enriched metal. Once the chelation sites have been filled, a maleimide moiety on the polymer is reacted with the free thiol groups on the partially reduced monoclonal immunoglobulin G (IgG) Ab to form an irreversible covalent bond. Here we describe modifications to the Fluidigm MaxPar® protocol that increase the quantity of Ab per reaction up to 150 μg, introduce an initial Ab quality control step, utilize metal labels not included in the Fluidigm catalog, and provide an option to perform two reactions in one centrifugal filter.

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Acknowledgments

This protocol was prepared with the support of the Ontario Institute for Cancer Research through funding provided by the Government of Ontario, The SickKids Centre for Advanced Single Cell Analysis (CASCA), and discussions with Fluidigm Corporation.

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Authors and Affiliations

  1. The Hospital for Sick Children Research Institute, Toronto, ON, Canada

    Stephen Gregory Chang & Cynthia J. Guidos

  2. Department of Immunology, University of Toronto, Toronto, ON, Canada

    Cynthia J. Guidos

Authors
  1. Stephen Gregory Chang
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  2. Cynthia J. Guidos
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Corresponding author

Correspondence to Stephen Gregory Chang .

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

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Chang, S.G., Guidos, C.J. (2019). Method for Tagging Antibodies with Metals for Mass Cytometry Experiments. 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_4

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  • DOI: https://doi.org/10.1007/978-1-4939-9454-0_4

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-4939-9453-3

  • Online ISBN: 978-1-4939-9454-0

  • eBook Packages: Springer Protocols

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