Mononuclear cells from porcine peripheral blood (PBMC) were isolated by density centrifugation of whole blood diluted 1:2 in 0.9% NaCl using Pancoll solution (density 1.077 g/mL, PAN-Biotech). PBMC were stained with the proliferation marker carboxyfluorescein diacetate succinimidyl ester (CFSE, eBiosciences) at a concentration of 5 mM for 5 min in the dark. CFSE-labeled porcine PBMCs were transferred to IMDM supplemented with 10% FCS and 1% penicillin–streptomycin (all PAN-Biotech, Aidenbach, Germany) and seeded into 96-well round bottom plates (2 Mio cells/200 μL). Proliferation was induced by adding Concanavalin A (ConA, 2 μg/mL; Sigma-Aldrich) and assessed after 5 days by comparing unstimulated and ConA-stimulated PBMC using flow cytometry.
For flow cytometry, cells were stained with the following antibodies specific to pig species: anti-CD4a-Pe-Cy7 (clone 4–12-4, IgG2b, BD Biosciences), anti-CD3ε-PerCP-Cy5.5 (clone BB23-8E6-8C8, IgG2a, BD Biosciences) and anti-CD8α-AlexaFluor® 647 (clone 76–2-11, IgG2a, BD Biosciences). For dead cell exclusion, a fixable viability dye was used in eFluor® 780 (eBiosciences). Cells were acquired on BD FACS Canto II with BD FACS Diva software and analyzed using FlowJo v9 software (Tree Star) for proliferative capacity of CD4+ T cells identified as liveCD3+CD4+CFSElow.
For flow cytometry, cells were stained with the following antibodies specific to pig species: anti-CD4a-Pe-Cy7 (clone 4–12-4, IgG2b, BD Biosciences), anti-CD3ε-PerCP-Cy5.5 (clone BB23-8E6-8C8, IgG2a, BD Biosciences) and anti-CD8α-AlexaFluor® 647 (clone 76–2-11, IgG2a, BD Biosciences). For dead cell exclusion, a fixable viability dye was used in eFluor® 780 (eBiosciences). Cells were acquired on BD FACS Canto II with BD FACS Diva software and analyzed using FlowJo v9 software (Tree Star) for proliferative capacity of CD4+ T cells identified as liveCD3+CD4+CFSElow.