Iron based ls columns

Blood samples were obtained from healthy human donors (Hospital Universitario 12 de Octubre, Madrid, Spain) subject to informed consent and IRB approval. Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll density gradient centrifugation (Ficoll Paque, GE17-5442-02, Sigma-Aldrich). To generate monocyte-derived dendritic cells (MDDCs), CD14+ monocytes were purified using anti-human CD14 antibody-labeled magnetic beads and iron-based LS columns (Miltenyi Biotec) and used directly for further differentiation into dendritic cells (Domínguez-Soto et al., 2011 (link)). Differentiation to immature MDDCs was achieved by incubation at 37°C with 5% CO2 for 7 days and subsequent activation with cytokines GM-CSF (1000 U/mL) and IL-4 (500 U/mL) (Miltenyi Biotec) every other day.

Blood samples were obtained from healthy human donors (Hospital 12 de Octubre, Madrid, Spain) under informed consent and IRB approval. Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll density gradient centrifugation (Ficoll Paque, GE17-5442-02, Sigma-Aldrich). To generate monocyte-derived dendritic cells (MDDCs) and monocyte-derived macrophages (M2-MDMs), CD14+ monocytes were purified using anti-human CD14 antibody-labeled magnetic beads and iron-based LS columns (Miltenyi Biotec) and used directly for further differentiation into dendritic cells and macrophages (Dominguez-Soto et al., J Immunol 2011). Differentiation to immature MDDCs was achieved by incubation at 37°C with 5% CO2 for 7 days and subsequent activation with cytokines GM-CSF (1000 U/mL) and IL-4 (500 U/mL) (Miltenyi Biotec) every other day. For differentiation of M2-MDMs, cells were incubated at 37°C with 5% CO2 for 7 days and activated with M-CSF (1000 U/mL) (Miltenyi Biotec) every second day.