Porcine Monocyte-Derived Dendritic Cell Generation

Porcine monocyte-derived dendritic cells (MoDCs) were generated as previously described [32 (link)]. Briefly, porcine monocytes were obtained from freshly collected porcine peripheral blood by a density gradient centrifugal method (1800 rpm, 20 min, 20 °C) with Lympholyte-mammal (Cedarlane, Hornby, ON, Canada). Cells were suspended in RPMI and plated (1 × 10⁷ cells/mL per well) into 12-well plates (Corning, Brumath, France) in RPMI-1640 supplemented with 2% FCS, 1% Streptomycin/Penicillin, and incubated two hours. Non-adherent cells were removed and remaining cells were incubated with RPMI supplemented with 10 μL/well of porcine GM-CSF (20 ng/mL) and 10 μL/well of porcine IL-4 (20 ng/mL). Fresh medium was renewed every 2 days. After 5 days of culture, cells were incubated with RPMI medium supplemented with GM-CSF, IL-4, and 5 μL/well of LPS (200 μg/mL) in order to induce differentiation of MoDC. The qRT-PCR was performed to quantify the expression of PGLYRPs mRNAs in porcine MoDCs. qRT-PCR was carried out with using Platinum SYBR Green qPCR SuperMix UDG with ROX (Invitrogen, Carlsbad, CA, USA). The primers used in this study are listed in Supplementary Table S2.