Isolation of Bovine Peripheral Blood Mononuclear Cells

Bovine venous whole blood was collected in sodium–heparin (25.000 I.U., ratiopharm, Ulm, Germany)-coated tubes, and PBMC were prepared as previously described [5 (link)]. Briefly, blood was diluted 1:2 in PBS (NaCl 136.9 mM (Sigma-Aldrich, part of Merck-Millipore, Darmstadt, Germany), Na₂HPO₄ × 2H₂O 8.1 mM (AppliChem, Darmstadt, Germany), KH₂PO₄ 1.4 mM (Merck-Millipore, Darmstadt, Germany), and KCl 2.6 mM (Sigma-Aldrich); pH 7.2), and PBMC were isolated by density gradient centrifugation (room temperature, 290× g, 25 min, brake off) using Pancoll separating solution (PanBiotech, Aidenbach, Germany). PBMC were obtained from the intermediate phase, washed twice in PBS, and immediately used for in vitro stimulation.