Anti human cd14 antibody conjugated microbeads

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.

Human monocytes were purified from patients or control. After partial purification via Ficoll-Hypaque centrifugation, monocytes were separated from lymphocytes by positive selection with anti-CD14. For positive selection of CD14⁺ monocytes, PBMCs were mixed with antihuman CD14 antibody-conjugated microbeads (Miltenyi Biotec, Auburn, CA, USA). CD14⁺ monocytes were separated from other cells using a magnetic separation column under a magnetic field (Miltenyi Biotec, Auburn, CA, USA) according to the manufacturer instructions and washed thoroughly to remove any non-specific binding to the beads. CD14⁺ cells were then eluted in PBS containing fetal-calf serum (FCS) (2%). Macrophages were obtained by culture of monocytes at 1×10⁶/ml in Teflon beakers in RPMI 1640 medium containing 15% human serum, 10 µg/ml gentamicin (Sigma-Aldrich) and 100 U/ml penicillin and 100 µg/ml streptomycin (Sigma-Aldrich) for 5–7 days.