Isolation and Characterization of Monocytes

MOs were isolated from PBMCs based on differential plastic adherence [27 (link)]. Briefly, PBMCs were cultivated in RPMI-1640 supplemented with 10% FCS and 50 μg/mL gentamicin, and seeded at 2 x 10⁶ cell/mL into 24-well plates. Cells were allowed to adhere for 2 h at 37°C before removal of non-adherent cells were and treatment of adherent MOs with MET. Cells were counted microscopically (Zeiss, Germany) using trypan blue staining, and the purity of monocytes was evaluated by fluorescent staining with PhycoErytherin (PE)-anti-human CD14 antibody (BD Biosciences, San Diego, CA, USA) using a Floid Cell Imaging Station (Thermo Fisher Scientific, MA USA) [28 (link),29 (link)] and routinely exceeded over 90% purity (S1 Fig).