Dendritic Cell Generation from PBMCs

DCs were prepared from fresh peripheral blood mononuclear cells (PBMCs) of healthy donors as described [21 (link)]. The donors gave informed consent to this experimental study, approved by the ethics committee of Guizhou Medical University. Highly enriched CD14⁺ monocytes were isolated from peripheral blood by Ficoll-Paque gradient centrifugation and purified by cocktail immunomagnetic beads (Dynal, Oslo, Norway). The monocytes were cultured in RPMI 1640 supplemented with 14% fetal bovine serum (FBS) (Gibco, Waltham, MA, USA), 1% penicillin/streptomycin, 150 ng/mL recombinant human GM-CSF (rhGM-CSF), and 100 ng/mL recombinant human IL-4 (rhIL-4) (Peprotech, Rocky Hill, NJ, USA). On day 7, 10 ng/mL recombinant human TNF-α (rhTNF-α) (Peprotech, Rocky Hill, NJ, USA) was added for another 72 h culture. The phenotypes of DCs were analyzed using flow cytometer (FACScan, Becton Dickinson, San Jose, CA, USA) by staining the cell surface with FITC- or PE-conjugated mouse anti-human CD11c, CD40, CD80, CD83, CD86, CCR7, and HLA-DR (Sigma-Aldrich, St. Louis, MO, USA). The trypan blue staining was applied to analyze the viability of cells as described [54 (link)].