A novel approach to study markers of dopamine signaling in peripheral immune cells

• DOI: 10.1016/j.jim.2019.112686
• Publication Date: 2019-10-18T16:01:03Z
• Authors: Adithya Gopinath; Andria Doty; Phillip M. Mackie; Basil Hashimi; Madison Francis; Leila Saadatpour; Kaustuv Saha; Gerry Shaw; Adolfo Ramirez-Za...; Michael S. Okun; Wolfgang J. Streit; Habibeh Khoshbouei
• Keywords: Adult; Cryopreservation; Male; Dopamine Plasma Membrane Transport Proteins; Tyrosine 3-Monooxygenase; Dopamine; Reproducibility of Results; Middle Aged; Flow Cytometry; Sensitivity and Specificity; 3. Good health; 03 medical and health sciences; 0302 clinical medicine; Vesicular Monoamine Transport Proteins; Leukocytes, Mononuclear; Humans; Female; Biomarkers; Aged; Signal Transduction

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.