Dendritic cell maturation

PBMCs were matured into dendritic cells using a published protocol [46]. PBMCs are cultured in dendritic base media. Briefly, AIM V cell culture media (Invitrogen) is supplemented with 10% human serum albumin (Gemini BioScience), β-mercaptoethanol (50μM), 1% Penicillin/Streptomycin, 0.1% amphotericin B, 800 U/mL GM-CSF (LifeTechmologies), 500 U/mL IL-4 (LifeTechnologies). After three days cells were fed with dendritic base media. On day 5 immature dendritic cells were fed with dendritic base media supplemented with 5 ng/mL TNF (Life Technologies), 5 ng/mL IL-1β (Life Technologies), 150 ng/mL IL-6 (Life Technologies), and 1 μg/mL prostaglandin E₂ (PGE₂; Life Technologies). On day 6 mature dendritic cells were shorted by fluorescence-activated cell sorting (FACs) for the expression of HLA-DR (Biolegend). On day 7 FACs sorted mature dendritic cells were co-cultured with control or H. pylori huFGOs.