Fluorescent target array killing assay: A multiplex cytotoxic T-cell assay to measure detailed T-cell antigen specificity and avidity in vivo

Authors

  • Benjamin J. C. Quah,

    Corresponding author
    1. Department of Immunology, John Curtin School of Medical Research, Australian National University, Canberra, ACT, 2601, Australia
    • Department of Immunology, John Curtin School of Medical Research, Building 131, Garran Road, The Australian National University, Canberra, ACT 2601, Australia
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  • Danushka K. Wijesundara,

    1. Department of Immunology, John Curtin School of Medical Research, Australian National University, Canberra, ACT, 2601, Australia
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  • Charani Ranasinghe,

    1. Department of Immunology, John Curtin School of Medical Research, Australian National University, Canberra, ACT, 2601, Australia
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  • Christopher R. Parish

    1. Department of Immunology, John Curtin School of Medical Research, Australian National University, Canberra, ACT, 2601, Australia
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Abstract

Here we describe a multiplex, fluorescence-based, in vivo cytotoxic T-cell assay using the three vital dyes carboxyfluorescein diacetate succinimidyl ester, cell trace violet, and cell proliferation dye efluor 670. When used to label cells in combination, these dyes can discriminate >200 different target cell populations in the one animal due to each target population having a unique fluorescence signature based on fluorescence intensity and the different emission wavelengths of the dyes. This allows the simultaneous measurement of the in vivo killing of target cells pulsed with numerous peptides at different concentrations and the inclusion of many replicates. This fluorescent target array killing assay can be used to measure the fine antigen specificity and avidity of polyclonal cytotoxic T-cell responses in vivo, immunological parameters that were previously impossible to monitor. © 2012 International Society for Advancement of Cytometry

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