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  • Identification and characterisation of transient receptor potential melastatin 2 and CD38 channels on natural killer cells using the novel application of flow cytometry

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    Author(s)
    Balinas, Cassandra
    Cabanas, Helene
    Staines, Donald
    Marshall-Gradisnik, Sonya
    Griffith University Author(s)
    Marshall-Gradisnik, Sonya M.
    Staines, Donald R.
    Year published
    2019
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    Abstract
    Background: Natural Killer (NK) cells are effector lymphocytes of the innate immune system and are subclassed into CD56BrightCD16Dim/− and CD56DimCD16+ NK cells. Intracellular calcium (Ca2+) is fundamental to regulate a number of intracellular signalling pathways and functions in NK cells, which are essential in mediating their natural cytotoxic function. Transient receptor potential melastatin 2 (TRPM2) is a Ca2+-permeable non-selective cation channel that possesses a critical role in calcium-dependent cell signalling to maintain cellular homeostasis. TRPM2 and CD38 protein surface expression has yet to be determined on ...
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    Background: Natural Killer (NK) cells are effector lymphocytes of the innate immune system and are subclassed into CD56BrightCD16Dim/− and CD56DimCD16+ NK cells. Intracellular calcium (Ca2+) is fundamental to regulate a number of intracellular signalling pathways and functions in NK cells, which are essential in mediating their natural cytotoxic function. Transient receptor potential melastatin 2 (TRPM2) is a Ca2+-permeable non-selective cation channel that possesses a critical role in calcium-dependent cell signalling to maintain cellular homeostasis. TRPM2 and CD38 protein surface expression has yet to be determined on NK cells using flow cytometry. Characterisation of TRPM2 has been previously identified by in vivo models, primarily using methods such as genetic remodification, immunohistochemistry and whole cell electrophysiology. The aim of this study was to develop an in vitro methodology to characterise TRPM2 and CD38 surface expression on NK cell subsets using an antibody that has not been previously applied using flow cytometry. Results: At 2 h/1 h, TRPM2 (Fig. 2 A, B, p < 0.05) and TRPM2/CD38 (Fig. 3A, B, p < 0.05) surface expression significantly increased between 1:300 and 1:50 at 2 h/1 h. TRPM2/CD38 surface expression furthermore increased between 1:100 and 1:50 at 2 h/1 h (Fig. 3A, p < 0.05). Interestingly, TRPM2/CD38 surface expression significantly decreased from 1:50 to 1:5 on CD56BrightCD16Dim/− NK cells. These consistent findings highlight that 1:50 is the optimal antibody dilution and threshold to measure TRPM2 and TRPM2/CD38 surface expression on NK subsets. 2 h/1 h was determined as the optimal incubation period to ensure a sufficient timeframe for maximal antibody binding and surface expression. Conclusion: For the first time, we describe an in vitro methodology to characterise TRPM2 and CD38 surface expression on NK cells in healthy participants. Finally, using an antibody that has not been previously applied in flow cytometry, we determined an antibody concentration and incubation time that is robust, rapid and sensitive for the application of flow cytometry.
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    Journal Title
    BMC Immunology
    Volume
    20
    Issue
    1
    DOI
    https://doi.org/10.1186/s12865-019-0293-0
    Copyright Statement
    © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License, which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.
    Subject
    Immunology
    Antibody
    Flow cytometry
    Natural killer cells
    Transient receptor potential Melastatin 2
    Publication URI
    http://hdl.handle.net/10072/386993
    Collection
    • Journal articles

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