Rosettesep human monocytes enrichment cocktail

Human moDCs were generated from monocytes as previously described (20 (link)). Monocytes were isolated from peripheral blood of 10 healthy individuals by negative selection using RosetteSep Human Monocytes enrichment cocktail (Stemcell Technologies, Vancouver, BC, Canada) according to manufacturer’s instructions. Monocytes were cultured at 2 × 10⁶ cells/ml in serum-free AIM-V medium (Gibco BLR, Grand Island, NY, USA), supplemented with 500 U/ml of recombinant human GM-CSF and IL-4 (eBioscience, San Diego, CA, USA) for 5 days at 37°C and 5% CO₂. At day 3, culture medium was replenished and cells were incubated with Dex (Sigma-Aldrich, St. Louis, CO, USA) at a final concentration of 1 µM [Dex-modulated DCs (D-DCs)]. At day 4, cells were stimulated with 1 µg/ml of cGMP-grade MPLA (Avanti Polar Lipids Inc., Alabaster, AL, USA) (DM-DCs). Unstimulated cells (DCs) and MPLA-matured DCs (M-DCs) generated in the absence of Dex were used as controls of immature and mature DCs, respectively. On day 5, cells were harvested and characterized by flow cytometry.

Human moDCs were generated from monocytes as previously described (10 (link)). Monocytes were isolated from peripheral blood by negative selection using RosetteSep Human Monocytes enrichment cocktail (Stemcell Technologies, Vancouver, Canada) according to manufacturer's instructions. Monocytes were cultured at 2 × 10⁶ cells/ml in serum-free AIM-V medium (Gibco BLR, Grand Island, NY, USA), supplemented with 500 U/ml of recombinant human GM-CSF and IL-4 (eBioscience, San Diego, CA, USA) for 5 days at 37°C and 5% CO₂. At day 3, culture medium was replenished and cells were incubated with dexamethasone (Sigma-Aldrich, St. Louis, CO, USA) at a final concentration of 1 μM. At day 4, cells were stimulated with 1 μg/ml of cGMP-grade MPLA (Avanti Polar Lipids Inc., Alabaster, AL, USA) (DM-DCs). Unstimulated cells (DCs) and MPLA-matured DCs (M-DCs) generated in the absence of dexamethasone were used as controls of immature and mature DCs, respectively. On day 5, cells were harvested and characterized by flow cytometry. Monocyte purity and gating strategy for DC characterization are shown on Supplementary Figure S1.