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.