Human pan monocyte isolation kit

PBMCs were isolated by standard density gradient centrifugation with Ficoll Paque Plus (GE Healthcare, 17-1440-03) from human buffy coats. Buffy coats were obtained from German Red Cross (Berlin, Germany) according to the current version of the declaration of Helsinki with an approved ethic vote (Charité, Berlin Germany; EA4/071/13). Untouched CD14⁺ and CD14⁺CD16⁺ monocytes were isolated by negative depletion using the human PAN monocyte isolation kit (Biolegend, 480060), following the manufacturer's instructions. Monocytes were cultured in commercial ready-to-use MoDC differentiation medium containing 400 IU/ml IL-4 and 500 IU/ml GM-CSF (Miltenyi Biotec, 130-094-812) at 37°C with 5% CO₂. Every two days, half of the culture medium was replaced with fresh medium. On day six, immature MoDCs were harvested, washed with PBS and resuspended in fresh medium at a density of 10⁶ cells/ml. Cells were either treated with medium only as control, 2.5 μg/ml LPS (from Escherichia coli O111:B4, L3024-5MG) or 400 μM NiSO4 (31483, tested endotoxin-free using QCL-1000TM Endpoint Chromogenic LAL Assay (Lonza, 50-647U) according to the manufacturers protocol). For proteomic studies and flow cytometry, cells were harvested after 24 h of continuous chemical incubation; for quantitative RT-PCR, cells were incubated either for 24 h or over a 36 h period with sample collections at various time points.