Heparin anticoagulant

Bovine peripheral blood mononuclear cells (PBMCs) were isolated from whole blood samples collected in 9 ml vacutainers containing Heparin anticoagulant (Greiner Bio-One, Kremsmünster, Austria). Whole blood was diluted 1:1 with HBSS and mixed gently before 25 ml was layered onto 15 ml of sterile Ficoll-paque density gradient medium (Fisher Scientific, New Hampshire, US) in a 50 ml tube. The samples were then centrifuged at 800×g for 25 mins with the centrifuge break turned off. The mononuclear cells were carefully removed with a pastette into a new 50 ml tube and made up to 10 ml with wash buffer (HBSS and 5% FBS). Cells were pelleted by centrifugation at 300×g for 5 mins. Any red blood cells carried over during the separation process were lysed by re-suspending the cells in red blood cell lysis buffer (Sigma-Aldrich). The cell pellet was then re-suspended in complete RPMI media (containing 10% FBS and 1% Pen-Strep) and cells were counted using a haemocytometer and trypan blue staining. Cell were then seeded at appropriate densities in tissue culture plates containing media. The cells were incubated at 37 °C with 5% CO₂.

Bovine peripheral blood mononuclear cells (PBMCs) were isolated from whole blood samples collected in 9 ml vacutainers containing Heparin anticoagulant (Greiner Bio-One, Kremsmünster, Austria). Whole blood was diluted 1:1 with HBSS and mixed gently before 25 ml was layered onto 15 ml of sterile Ficoll-paque density gradient medium (Fisher Scientific, New Hampshire, US) in a 50 ml tube. The samples were then centrifuged at 800 × g for 25 min with the centrifuge break turned off. The mononuclear cells were carefully removed with a pastette into a new 50 ml tube and made up to 10 ml with wash buffer (HBSS and 5% FBS). Cells were pelleted by centrifugation at 300 × g for 5 min. Any red blood cells carried over during the separation process were lysed by re-suspending the cells in red blood cell lysis buffer (Sigma-Aldrich). The cell pellet was then re-suspended in complete RPMI media (containing 10% FBS and 1% Pen-Strep) and cells were counted using a haemocytometer and trypan blue staining. Cell were then seeded at appropriate densities in tissue culture plates containing media. The cells were incubated at 37°C with 5% CO₂.