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.