PBMC isolation, culture, and treatment
Mutual interaction of microglia and immune cells is indicated to be present in depression. Therefore concerning microglia’s central role in the development of depressive behavior, studies represented MDD as a microgliopathy 20. Microglia are the present immune cells of the central nervous system and despite some differences, they share a similar progeny with peripheral immune cells. Given their similar progeny, microglia-like cells have been induced from the human peripheral blood monocyte cells in culture 21,22. Therefore, PBMCs response could reflect the drug impact on both peripheral and microglia in patients as previously indicated23. Furthermore, according to the literature inflammatory status of the monocytes in MDD patients determines the response rates to some antidepressants 1. Therefore, we chose the PBMCs model to explore bupropion immunomodulatory effects. PBMCs were isolated from the whole blood collected from 5 healthy volunteers via Ficoll (Baharafshan, Iran) gradient centrifugation and washed three times subsequently with RPMI-1640 medium (BioIdea, Iran). Cells were suspended in RPMI 1640 medium thereafter (supplemented with 100 U of penicillin/ml, 100 μg/ml streptomycin, 2 mM L-glutamine, and 10% Fetal Bovine Serum FBS) (FBS, Biochrom, Berlin, Germany). The cell viability was evaluated using trypan blue (Innoclon, Iran), and cells with a viability of more than 95% were selected for culture. Cells were transferred in duplicate into the wells of a 96-well microplate at the densities of 104 cells/well. The microculture plate was incubated at 37ºC under 5% CO2 with 90% humidity. To induce cell proliferation, phytohemagglutinin (10 μg/ml) (PHA, Sigma-Aldrich, USA) was added to the wells in the presence or absence of bupropion. LPS (100 ng/ml) was also added to the test culture and incubated at 37°C, 5% CO2, for 24 hours. Finally, bupropion was administered to PBMCs at different concentrations (25, 50, 100, 200 μM). Following 48‐hour incubation, plates were centrifuged at 1700 RPM for 10 minutes, and Supernatants get collected.