In vitro Neutrophil Priming Assay

In vitro stimulation of purified blood neutrophils was performed based on a previously published method for bovine neutrophils with minor changes [20 (link),23 (link)]. Isolated neutrophils were suspended in HBSS at a final concentration of 1 × 10⁷ cells/mL. For the in vitro priming of neutrophils, 1 × 10⁶ cells in 100 µL medium were incubated for 30 min at 37 °C and 5% CO2 with 1 μg/mL of the TLR4-ligand LPS, 1 μg/mL of the TLR2/1-ligand Pam3CSK4, 0.2 µg/mL of the TLR7/8-ligand R848 (Resiquimod), 10 µg/mL of the TLR3-ligand Poly IC, or 10 ng/mL of the protein kinase C activator phorbol 12-myristate 13-acetate (PMA), or only HBSS medium without stimulants. The concentrations of the stimulants were chosen based on previously described concentrations for the stimulation of bovine cells [20 (link)].