Isolation of peripheral human CD14+monocytes and in vitro experiments
Peripheral blood mononuclear cells were isolated from a healthy adult donor using Ficoll–Hypaque density gradient centrifugation. CD14+ monocytes (anti-CD14 microbeads, MACS, Miltenyi Biotec, Bergisch Gladbach, Germany) were separated from the peripheral blood mononuclear cells using the OctoMACS separator and starting kits (MACS, Miltenyi Biotec) (Park et al., 2020). Separated human CD14+ monocytes were plated in 90 × 20-mm cell culture dishes (SPL Life Sciences, Pochon, Korea) at a density of 1 × 106 cells/mL in serum-free Roswell Park Memorial Institute 1640 medium. The medium was pre-treated with TAF for 6 h. The control group was treated with an equal volume of dimethyl sulfoxide (DMSO). The medium was refreshed with 1 μg/mL LPS or mock for 24 h. The cells were then incubated at 37 °C with 5% CO2. We then performed fluorescence staining (AKT, CD45, human leukocyte antigen-DR isotype [HLA-DR], phospho-AKT, programmed death-ligand 1 [PD-L1], and LIVE/DEAD dye). The research protocol conformed to the Declaration of Helsinki; the institutional review board of St. Mary’s Hospital in Seoul (KC20TISI0817) approved the study. Informed consent was obtained from all the participants.