Peripheral blood mononuclear cells (PBMCs) were isolated by centrifugation on a density gradient (Lymphoprep, 07801, StemCell Technologies) following the manufacturer's protocol. Primary blood DCs were purified according to an established protocol (Alculumbre and Pattarani, 2016 (link)). In brief, total PBMCs were enriched in DCs using the EasySep Human Pan-DC Pre-Enrichment kit (StemCell Technologies). Enriched DCs were then sorted to obtain 98% purity on an MoFlo Astrios cell sorter (Beckman Coulter) or FACS ARIA III (BD Technologies), as lineage− (CD3, CD14, CD16, CD56, CD20 and CD19; FITC anti-human CD3e, BD, 555339; FITC anti-human CD14, Miltenyi Biotec, 130-080-701; FITC anti-human CD16, BD, 347523; FITC anti-human CD56, BioLegend, 318304; FITC anti-human CD20, BD, 555622; FITC anti-human CD19, Miltenyi Biotec, 130-113-168), CD4+ (BV-650 anti-human CD4, BioLegend, 317436), CD11c+ (PeCy7 anti-human CD11c, Biolegend, 337216) and CD1c+ (PerCP-eFluor710 anti-human CD1c, also known as BDCA1, Thermo Fisher Scientific, 46-0015-42).
After enrichment from total PBMCs using the CD4+ T cell isolation kit (StemCell Technologies), naive CD4+ T cells were magnetically isolated. Purity was at least 95%.
After enrichment from total PBMCs using the CD4+ T cell isolation kit (StemCell Technologies), naive CD4+ T cells were magnetically isolated. Purity was at least 95%.