4.4. Isolation of Mononuclear Cells from Blood and Differentiation of Human Macrophages

Human macrophages were differentiated from peripheral blood monocytes. Monocytes were isolated from healthy donors using Ficoll-Paque Premium (GE Healthcare, Chicago, IL, USA) and the adherence method [62]. Monocytes were incubated in RPMI-1640 medium supplemented with 2% heat-inactivated human AB serum, 2 mM L-glutamine, 10 mM HEPES, 50 µM β-mercaptoethanol, 2 mM sodium pyruvate, and 2 mM MEM Vitamin (HyClone, Uhta, UT, USA) at 37 °C and 5% CO₂ for 6 days. On the 4th day, the medium was fully refreshed. On the first and fourth days, 50 ng/mL GM-CSF (Sci-Store, Moscow, Russia) was added to the medium. Cells were stained with fluorophore-conjugated primary antibodies against CD11b (APC-Cy7), CD80 (PE-Cy5), CD86 (BV421), HLA-DR (PE-Cy7) and analyzed by a flow cytometer (Navios, Beckman Coulter, Inc., Brea, CA, USA).