Abstract
BackgroundNatural 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.ResultsAt 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.ConclusionFor 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.
Highlights
Natural Killer (NK) cells are effector lymphocytes of the innate immune system and are subclassed into CD56BrightCD16Dim/− and CD56DimCD16+ NK cells
Immunophenotype of Transient receptor potential melastatin 2 (TRPM2) and CD38 receptors on NK cell subsets by flow cytometry CD3−/CD56+ NK cells were sorted into CD56DimCD16/+ and CD56BrightCD16Dim/− NK cell subsets using CD56 (Pe-Cy7) and CD16 (BV650)
Antibody controls included an unstained tube; secondary tube; and a Fluorescence minus one (FMO) tube (CD3, CD56, CD16 and CD38). (b) Normal rabbit serum was used at comparable dilutions as the primary TRPM2 antibody to measure TRPM2 and TRPM2/CD38 surface expression on NK subsets. (c) Normalised TRPM2 and TRPM2/CD38 surface expression was calculated by compensating the percentage of fluorescence spill over into the B525_50 (TRPM2) and V525_50 (CD38) detectors from the TRPM2 antibody stained tube on both NK subsets
Summary
Natural Killer (NK) cells are effector lymphocytes of the innate immune system and are subclassed into CD56BrightCD16Dim/− and CD56DimCD16+ NK cells. NK cells have diverse biological functions, which include recognizing and killing virally infected or transformed cells The former NK population is primarily involved in immunosurveillance and cytokine production, whereas the latter are cytotoxic and kill infected, tumour or ‘missing self’ cells [2]. Intracellular calcium (Ca2+) mobilisation is required to regulate a number of intracellular signalling pathways in NK cells, such as the antibody dependent cellular cytotoxicity (ADCC) or mitogen-activated protein kinase pathway, which are essential for the development of immune synapse formation, cytokine production and cytotoxic activity [1]. Intracellular Ca2+ is required for the target cell adhesion, granule polarization and degranulation, all of which are necessary for NK cells to mediate their natural cytotoxicity [1, 3]
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