Human monocytes express known markers of dopamine synthesis, storage and clearance, including dopamine transporter (DAT), tyrosine hydroxylase (TH), all subtypes of dopamine receptors and vesicular monoamine transporter 2 (VMAT2). Immunohistochemical and immunofluorescent methodologies have traditionally been employed to determine DAT and TH expression in the CNS, their detection in the blood and specifically in the peripheral monocytes has not been studied by flow cytometry. Flow cytometry assays are widely used in medicine and in basic, preclinical or clinical research to quantify physical and chemical characteristics of target cell populations. Here, we have established a highly sensitive and reproducible flow cytometry panel to detect and quantify DAT and TH expression in freshly isolated or cryopreserved human peripheral monocytes. In healthy humans (n = 41 biological replicates), we show baseline DAT and TH expressing monocytes constitute ~12% of the peripheral blood mononuclear cell (PBMC) fraction when examined in fresh isolation from whole blood. Using an identical flow cytometry panel, we found that cryopreservation of PBMCs using multiple techniques resulted in altered PBMC populations as compared to fresh isolation and relative to one another. Among these, we identified an optimum cryopreservation method for detecting TH and DAT in cryopreserved PBMCs. Our data provide a sensitive and reproducible approach to examine dopamine signaling in peripheral human immune cells. This approach can be applied to study peripheral dopamine signaling under healthy and potentially under disease conditions. The use of dopamine signaling could also be explored as a technique to monitor therapeutic interventions particularly those targeting DAT and TH in the periphery.

中文翻译：

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一种研究外周免疫细胞中多巴胺信号传导标记的新颖方法。

人单核细胞表达多巴胺合成，储存和清除的已知标记，包括多巴胺转运蛋白（DAT），酪氨酸羟化酶（TH），多巴胺受体的所有亚型和囊泡单胺转运蛋白2（VMAT2）。免疫组织化学和免疫荧光方法传统上已用于确定CNS中DAT和TH的表达，尚未通过流式细胞术研究其在血液中的检测，尤其是在外周单核细胞中的检测。流式细胞仪测定法广泛用于医学和基础，临床前或临床研究中，以量化靶细胞群的物理和化学特征。在这里，我们建立了一个高灵敏度和可再现的流式细胞仪面板，以检测和量化新鲜分离或冷冻保存的人外周血单核细胞中的DAT和TH表达。在健康的人类中（n = 41个生物学重复），当从全血中新鲜分离出来进行检查时，我们显示基线DAT和TH表达的单核细胞约占外周血单核细胞（PBMC）比例的12％。使用相同的流式细胞仪面板，我们发现与新鲜分离相比，使用多种技术对PBMC进行冷冻保存会导致PBMC群体发生变化。在这些方法中，我们确定了一种最佳的冷冻保存方法，用于检测冷冻保存的PBMC中的TH和DAT。我们的数据提供了一种灵敏且可重现的方法来检查外周人类免疫细胞中的多巴胺信号传导。该方法可用于研究健康以及潜在疾病条件下的外周多巴胺信号传导。