Isolation of primary human and bovine monocytes and differentiation into macrophages

Human monocytes were obtained from leukocyte enriched blood fractions from healthy adult donors provided by the British National Health Service (NHS) under strict anonymity. Bovine blood was obtained from clinically healthy Holstein cows in mid-lactation phase (with a somatic cell count below 100,000 as an indicator of healthy udders) housed at Bolton Park’s Farm of the Royal Veterinary College (University of London). All animals were regularly tested for freedom of TB infection (as per governmental guidelines) and were in addition free of Bovine Viral Diarrhoea Virus (BVDV) and Infectious Bovine Rhinotracheitis (IBR), both viral infections leading to immunosuppression. Blood was collected by jugular venepuncture into sterile glass bottles containing 10% Acid Citrate Dextrose (ACD) as anticoagulant, as previously described [74]. Prior to monocyte isolation, bovine whole blood was aliquoted into 50 mL centrifugation tubes and centrifuged for 15 min at 1200 x g to collect the buffy coat. Both human leucocyte enriched fraction and bovine buffy coat were then processed in the standard way but with the difference that PBS-EDTA was used as the buffer for the isolation of human monocytes, while PBS was used for the processing of bovine blood. Peripheral Blood Monocytes Cells (PBMCs) were isolated by centrifugation onto a Ficoll gradient. PBMCs were then washed and CD14-expressing monocytes were labelled with the anti-human CD14-antibody conjugated with magnetic beads (Miltenyi #130-050-201) and isolated by direct magnetic selection using the LS column (Miltenyi #130-042-401). Human and bovine CD14-positive monocytes were finally incubated in RPMI 1640 medium (Gibco # 72400021) containing: GlutaMax, 25 mM Hepes, 10% Foetal bovine serum (FBS) and 10 ng/mL of recombinant human GM-CSF or 20 ng/mL of recombinant bovine GM-CSF to allow differentiation into Mϕ for 7 days at 37°C in an atmosphere containing 5% CO₂.