2.1.2. Flow Cytometry Analysis
Human peripheral blood mononuclear cells (PBMCs) from healthy donors were purchased from iQ Biosciences (Alameda, CA, USA) and Lonza (Basel, Switzerland). PBMCs were cultured in a RPMI 1640 medium (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific), 100 U/mL penicillin, and 100 μg/mL streptomycin (Thermo Fisher Scientific) at 37 °C and 5% COPBMCs were seeded and treated with 10 μg/mL LG2055 for 24 h at 37 °C and 5% COFluorescence was measured using a FACSCanto II flow cytometer (BD), and the data were analyzed using FACSDiva software version 6.1.3 (BD). In this assay, live Lineage (Lin1)− HLA-DR+ CD123+ CD11c− cells were defined as pDCs, while live Lin1− HLA-DR+ CD123− CD11c+ cells were defined as myeloid dendritic cells (mDCs), and both groups of cells were analyzed. The expression levels of cell surface markers (CD86 and HLA-DR) represented pDC and mDC activity. The mean fluorescence intensity (MFI) was measured and used for the analyses. Each experiment was performed in triplicate.