Magnetic cell sorting monocyte isolation kit 2

By using Ficoll density gradient centrifugation (Amersham, Freiburg, Germany), human cord blood mononuclear cells (CBMC) as well as human peripheral blood mononuclear cells (PBMC) were isolated. Afterwards, the cells were washed with PBS, and monocytes were separated from remaining cell types by using the magnetic cell sorting monocyte isolation kit II (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer's recommendation. Detected by flow cytometry, the method routinely yielded 95% purity of the population while 90% CD14-positive cells were defined as minimal cut-off value. The cells were standardly cultivated in VLE RPMI-1640 medium (Biochrom, Berlin, Germany) containing 10% heat-inactivated fetal bovine serum (FBS, Biochrom, Germany) and 1% penicillin/streptomycin (Thermo Fisher, Massachusetts, USA). Postphagocytic reaction experiments were performed in 24-well cell culture plates (Costar, Bodenheim, Germany) containing 1 × 10⁶ monocytes/ml.

Isolation of peripheral blood mononuclear cells (PBMC) and cord blood mononuclear cells (CBMC) was performed by using Ficoll density gradient centrifugation (Amersham, Freiburg, Germany). Cells were washed and resuspended in VLE-RPMI 1640 (Biochrom, Berlin, Germany). To separate the monocytes, magnetic cell sorting monocyte isolation kit II (Miltenyi Biotec, Bergisch Gladbach, Germany) was used according to the manufacturer's protocol. The minimal purity of the resulting population was defined to be 90% CD14-positive cells, as detected by flow cytometry. For analysis of postphagocytic reactions, 10⁶ cells/ml were seeded in 24-well cell culture plates (Costar, Bodenheim, Germany) in VLE-RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS, Biochrom, Germany) and 1% penicillin/streptomycin (PenStrep, Thermo Fischer, Massachusetts, USA).