Generation of Porcine Monocyte-Derived Dendritic Cells

Monocytes were separated from PBMCs by plastic adherence for 90 min at 37 °C in 5% CO₂. Afterwards, PBLs were removed and immediately frozen for long-term storage at −150 °C as described by Leitner et al. [23 (link)]. The remaining plastic-adherent cells were washed twice with CM. MoDCs were generated as previously described by Carrasco et al. [24 (link)] with minor modifications. Briefly, plastic-adherent monocytes were cultured in CM supplemented with 40 ng/mL of recombinant porcine (rp) GM-CSF (R&D Systems, Minneapolis, MN, USA) and 40 ng/mL of rpIL-4 (R&D Systems) at 37 °C in 5% CO₂. After three days the medium was replaced by fresh cytokine-supplemented CM. Seven days after the start of in vitro cultivation moDCs were harvested with a cell scraper (Greiner Bio-One).