Bovine Monocyte-derived Macrophage Protocol

BoMØs were generated following the protocol described by García-Sánchez et al. (2019 (link)). Briefly, 900 ml of peripheral blood was collected from a healthy adult Holstein dairy cow that tested negative for infectious bovine rhinotracheitis virus (IBRV), bovine viral diarrhea virus (BVDV) and N. caninum. Histopaque 1077 (Sigma-Aldrich, USA) was used to separate peripheral blood mononuclear cells by density gradient, and isolation of monocytes was carried out by positive selection using anti-human CD14 antibody-conjugated microbeads (Miltenyi Biotec Ltd., USA). Monocytes were incubated at 37°C in a 5% CO₂ atmosphere in medium containing 100 ng/ml recombinant bovine GM-CSF (Kingfisher Biotech Inc, USA). At day 5, boMØs were harvested, reseeded at 3 × 10⁶ cells/well in 6-well culture plates (P6) and incubated for 24 h prior to infection to minimize cellular stress due to the harvest procedure.