Rpil 4

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).

Alveolar Macrophages (AMs) and PBMCs were obtained from the same pigs as described in “Obtention of Bone Marrow Cells” through, respectively, broncho-alveolar lavage and density gradient centrifugation with Histopaque 1.077 (Sigma-Aldrich, Spain). AMs and PBMCs were frozen in liquid nitrogen until used. The selected batches had viabilities >90% after thawing as assessed by trypan blue staining. For PBMCs, the proportion of each T cell subset and the proliferation capability were also tested in one sample per batch. Batches passing this quality control (viability and proliferation capability) were used. Monocytes were obtained through plastic adherence of PBMCs for 16 h at 37°C in 5% CO₂ and used freshly. moDCs were generated by differentiating blood monocytes with recombinant porcine GM-CSF (rpGM-CSF, 20 ng/ml; R&D Systems, Spain) and rpIL-4 (20 ng/ml; R&D Systems, Spain) according to the method described by Carrasco et al. (16 (link)).