Lymphocyte separation medium

The local ethics committee at the University Medical Center approved this study (approval number: BB166/17). Human peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats of healthy donors provided by the Institute of Transfusion Medicine Greifswald by density-gradient centrifugation using lymphocyte separation medium (VWR, Germany). Residual erythrocytes were lysed using a red blood cell (RBC) lysis buffer (Thermo Fisher, Germany). For monocyte isolation, CD14 microbeads (Miltenyi Biotec, Germany) were used according to the manufacturer's protocol. Flow cytometric (Attune NxT; Applied Biosystems, USA) verification of purity was performed immediately after isolation and was always greater than 85%. 1 × 10⁵ monocytes per well were seeded in 24-well plates (Sarstedt, Germany). The cell culture medium was supplemented with 800 IU granulocyte–macrophage colony-stimulating factor (GM-CSF) and 500 IU interleukin (IL) 4 (all PeproTech, Germany) to initiate monocyte-derived dendritic cells (DCs). After two days, cells were re-stimulated by adding the same amounts of GM-CSF and IL-4 to the cell culture medium. Immature DCs were used for co-culture experiments on day 5.

Isolation of PBMCs in this study was approved by the local ethics committee at the University Medical Center (approval number: BB 014‐14). PBMCs were isolated from buffy coats of healthy donors provided by the Institute of Transfusion Medicine Greifswald by density‐gradient centrifugation using lymphocyte separation medium (VWR, Germany). Residual erythrocytes were lysed using a red blood cell lysis buffer (BioLegend, Germany). For monocyte isolation, CD14 microbeads (Miltenyi Biotec, Germany) were used according to the manufacturer's protocol. Flow cytometric (Attune NxT; Applied Biosystems, USA) verification of purity was performed immediately after isolation and was always greater than 85%. For differentiation of monocytes into DCs, 1 × 10⁵ monocytes per well were seeded in 24‐well plates (Sarstedt, Germany). The cell culture medium was supplemented with 800 IU granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) and 500 IU interleukin (IL) 4 (all PeproTech, Germany) to initiate monocyte‐derived dendritic cells (DCs). After two days, cells were restimulated by adding the same amount of GM‐CSF and IL‐4 to the cell culture medium. Immature DCs were used for coculture experiments on day 5.