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Automation of sample preparation for mass cytometry barcoding in support of clinical research: protocol optimization

Analytical and Bioanalytical Chemistry volume 409, pages 2363–2372 (2017)Cite this article

Abstract

Analysis of multiplexed assays is highly important for clinical diagnostics and other analytical applications. Mass cytometry enables multi-dimensional, single-cell analysis of cell type and state. In mass cytometry, the rare earth metals used as reporters on antibodies allow determination of marker expression in individual cells. Barcode-based bioassays for CyTOF are able to encode and decode for different experimental conditions or samples within the same experiment, facilitating progress in producing straightforward and consistent results. Herein, an integrated protocol for automated sample preparation for barcoding used in conjunction with mass cytometry for clinical bioanalysis samples is described; we offer results of our work with barcoding protocol optimization. In addition, we present some points to be considered in order to minimize the variability of quantitative mass cytometry measurements. For example, we discuss the importance of having multiple populations during titration of the antibodies and effect of storage and shipping of labelled samples on the stability of staining for purposes of CyTOF analysis. Data quality is not affected when labelled samples are stored either frozen or at 4 °C and used within 10 days; we observed that cell loss is greater if cells are washed with deionized water prior to shipment or are shipped in lower concentration. Once the labelled samples for CyTOF are suspended in deionized water, the analysis should be performed expeditiously, preferably within the first hour. Damage can be minimized if the cells are resuspended in phosphate-buffered saline (PBS) rather than deionized water while waiting for data acquisition.

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Abbreviations

CyTOF:

Cytometry by Time-Of-Flight

FB:

Fixation buffer

ICP-MS:

Inductively coupled plasma-mass spectrometry

MCB:

Mass-tag cellular barcoding

PBMC:

Peripheral blood mononuclear cell

PMA:

Phorbol 12-myristate 13-acetate, also known as 12-O-tetradecanoylphorbol-13-acetate (TPA)

SB:

Maxpar Cell Staining Buffer

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Acknowledgements

This work is supported in part by a grant from Centers for Disease Control and Prevention/The National Institute for Occupational Safety and Health (OH110941). The authors thank the members of the Yale CyTOF user’s group for helpful discussions.

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Affiliations

  1. School of Medicine, Department of Internal Medicine, Yale University, 300 Cedar St./TAC s416, New Haven, CT, 06510, USA

    Ala F. Nassar & Adam V. Wisnewski

  2. Department of Chemistry, University of Connecticut, 55 North Eagleville Road, Storrs, CT, 06269, USA

    Ala F. Nassar

  3. Department of Neurology, Yale School of Medicine, New Haven, CT, 06511, USA

    Khadir Raddassi

Authors
  1. Ala F. Nassar
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  2. Adam V. Wisnewski
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  3. Khadir Raddassi
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Correspondence to Ala F. Nassar.

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Nassar, A.F., Wisnewski, A.V. & Raddassi, K. Automation of sample preparation for mass cytometry barcoding in support of clinical research: protocol optimization. Anal Bioanal Chem 409, 2363–2372 (2017). https://doi.org/10.1007/s00216-017-0182-4

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  • DOI: https://doi.org/10.1007/s00216-017-0182-4

Keywords

  • Multiplexing
  • Barcoding
  • CyTOF
  • Mass cytometry