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.

中文翻译：

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使用流式细胞仪的新应用鉴定和表征自然杀伤细胞上的瞬时受体电位褪黑素2和CD38通道。

背景技术天然杀伤（NK）细胞是先天免疫系统的效应淋巴细胞，并被亚分类为CD56BrightCD16Dim /-和CD56DimCD16 + NK细胞。细胞内钙（Ca2 +）是调节NK细胞中许多细胞内信号传导途径和功能的基础，这在介导其天然细胞毒性功能中至关重要。瞬时受体电位褪黑素2（TRPM2）是可透过Ca2 +的非选择性阳离子通道，在钙依赖性细胞信号传导中维持细胞稳态具有关键作用。尚未使用流式细胞术确定NK细胞上TRPM2和CD38蛋白的表面表达。先前已通过体内模型确定了TRPM2的特性，主要使用诸如遗传修饰，免疫组织化学和全细胞电生理学等方法。这项研究的目的是开发一种体外方法，使用以前未使用流式细胞仪测定的抗体来表征NK细胞亚群上TRPM2和CD38表面的表达。结果在2 h / 1 h时，TRPM2（图2 A，B，p <0.05）和TRPM2 / CD38（图3A，B，p <0.05）在2：1到1:50之间的表面表达显着增加。小时/ 1小时。在2 h / 1 h时，TRPM2 / CD38表面表达在1：100和1:50之间进一步增加（图3A，p <0.05）。有趣的是，在CD56BrightCD16Dim /-NK细胞上，TRPM2 / CD38表面表达从1:50显着降低至1：5。这些一致的发现表明，1:50是测量NK亚群上TRPM2和TRPM2 / CD38表面表达的最佳抗体稀释度和阈值。确定2 h / 1 h为最佳孵育时间，以确保有足够的时间框架实现最大抗体结合和表面表达。结论我们首次描述了体外方法表征健康参与者NK细胞上TRPM2和CD38表面表达的特征。最后，使用之前未在流式细胞术中应用的抗体，我们确定了对于流式细胞术的应用而言可靠，快速且灵敏的抗体浓度和孵育时间。