A novel approach to study markers of dopamine signaling in peripheral immune cells.
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.
['Gopinath|Adithya|A|', 'Doty|Andria|A|', 'Mackie|Phillip M|PM|', 'Hashimi|Basil|B|', 'Francis|Madison|M|', 'Saadatpour|Leila|L|', 'Saha|Kaustuv|K|', 'Shaw|Gerry|G|', 'Ramirez-Zamora|Adolfo|A|', 'Okun|Michael S|MS|', 'Streit|Wolfgang J|WJ|', 'Khoshbouei|Habibeh|H|']
D000328:Adult D000368:Aged D015415:Biomarkers D015925:Cryopreservation D004298:Dopamine D050483:Dopamine Plasma Membrane Transport Proteins D005260:Female D005434:Flow Cytometry D006801:Humans D007963:Leukocytes, Mononuclear D008297:Male D008875:Middle Aged D015203:Reproducibility of Results D012680:Sensitivity and Specificity D015398:Signal Transduction D014446:Tyrosine 3-Monooxygenase D050493:Vesicular Monoamine Transport Proteins
Peripheral immune cells Human monocytes Dopamine transporter Tyrosine hydroxylase Flow cytometry Peripheral blood mononuclear cell