Lymphosep

Crossbred, PRRSV-seronegative pigs were previously immunized with CSFV-modified live vaccine (MLV) (COGLAPEST®, Ceva Santé Animale, Libourne, France) at 4 and 7 weeks of age. At 16 weeks of age, porcine peripheral blood mononuclear cells (PBMC) were isolated from heparinized whole blood by density gradient centrifugation, using LymphoSep™ (MP Biomedicals, California, USA) according to the manufacturer's procedure. MoDC were generated as previously described (34 (link)). Briefly, the PBMC were resuspended at 5 x 10⁶ cells/mL in Iscove's Modified Dulbecco's Media (IMDM) (GIBCO, Carlsbad, CA, USA), and incubated at 37°C and 5% CO₂ for 2 h. Non-adherent cells, referred as peripheral blood lymphocytes (PBL), were collected and stored at 5 × 10⁷ cells/mL in liquid nitrogen until needed. The remaining adherent cells were cultured with 10 ng/mL porcine recombinant IL-4 (R&D system, Minneapolis, MN, USA) and 25 ng/mL porcine recombinant GM-CSF (R&D system) for 7 days. For downstream experiments, PBMC, PBL, and MoDC were plated in complete RPMI, containing advanced RPMI (GIBCO), 10% FBS (GIBCO), 2 mM L-glutamine (GIBCO), antibiotic/antimycotic solution (GIBCO), 25 mM HEPES (GIBCO), and 50 μM β-mercaptoethanol (Sigma Chemical Co., St. Loius, USA).

PBMCs were isolated from 9 ml samples of whole blood using LYMPHOSEP™ (MP Biomedicals LLC, Ohio, USA), according to the manufacturer's instructions. The obtained pellet was suspended in 4 ml of a Lymphogrow medium (Cytogen-Polska Sp. z o.o., Zgierz, Poland) containing phytohemagglutinin (PHA) and recombinant IL-2 (4 ng, 100 U, BioLegend, San Diego, CA, USA). The suspension was then transferred to a 25-ml vessel for adherent culture. Cells were grown under standard conditions at 37°C, 5% CO₂ with shaking for 24 h. Non-adherent cells were washed with PBS and transferred to a 25-ml vessel for suspension culture with fresh Lymphogrow medium supplemented with IL-2. After another 48 h, the cells were passaged and maintained in a culture using a standard RPMI-1630 medium supplemented with L-glutamine (2 mM), FBS (10%), penicillin (100 IU/ml), streptomycin (100 μg/ml), and with the addition of IL-2. Cell differentiation was measured by CD3, CD4, CD8, CD45, and HLA-DR by flow cytometry analysis. In the third passage, anti-TNF mAbs' (Sigma) was added (10 μg/ml). In parallel, a control culture without the addition of the antibody was carried out. After 72 h of culture, cells were suspended in 200 μl stayRNA solution (A&A Biotechnology, Gdansk, Poland) and frozen at −80°C for RNA isolation. Moreover, part of the cells was subjected to apoptosis analysis.