2.5. Generation of Monocyte-Derived Macrophages (MDM) and Monocyte-Derived Dendritic Cells (MoDC)

Human monocytes were purified from heparinized blood samples derived from CD, UC or healthy subjects by Ficoll density gradient separation and by positive selection using CD14⁺ selection (CD14 Microbeads, Miltenyi Biotec, Bergisch Gladbach; Germany).

For generation of monocyte-derived macrophages (MDM), CD14⁺ cells were seeded into flat bottom 96-well culture plates at a density of 2 × 10⁵ cells/well in complete RPMI medium supplemented with 100 ng/mL of recombinant human macrophage-colony stimulating factor (rh-M-CSF, Miltenyi Biotec). The cells were incubated in a humidified atmosphere at 37 °C with 5% CO₂. After 2 and 4 days, all the unattached cells were discarded by removing half of the culture media and replacing it with complete RPMI with twice the final concentration of M-CSF. On day 7 the total number of MDM cells per well, as well as the morphology and the expected immunophenotype, were confirmed by flow cytometry (data not shown).

For generation of monocyte-derived dendritic cells (MoDC), CD14⁺ cells were cultured into 24-well culture plates at a density of 10⁶ cells/mL in complete RPMI medium supplemented with 100 ng/mL of recombinant human granulocyte–monocyte colony stimulating factor (rh-GM-CSF, Miltenyi Biotec) and 50 ng/mL of Interleukin-4 (IL-4, Miltenyi Biotec). After 3 days, culture media were replaced by removing half of the complete medium and replacing it with complete RPMI with twice the final concentration of cytokines used for differentiation. Thereafter, MoDC were collected and seeded into round bottom 96-well culture plates at a density of 10⁵ cells/well in complete RPMI medium without pen/strep for the coinfection experiments. The morphology, as well as the immunophenotype analysis to check for proper differentiation of monocytes into MoDC, were confirmed at this time point by flow cytometry before the infection experiments (data not shown).