Chemotaxis assay

Chemotaxis assay was conducted using a 96-well filter plate with 5 μm pore size polycarbonate membrane (MAMIC 5S10, Millipore, Billerica, MA, USA). Then, 5 μM fMLP (Sigma-Aldrich, St. Louis, MO, USA) was diluted in 27 μL HBSS (without calcium and magnesium) containing 0.5% BSA and placed in the lower chamber. Neutrophils (50 μL; 2 × 10⁵/mL) from mouse bone marrow diluted in the same medium were loaded into the upper wells after being incubated with compounds at the indicated concentrations for 30 min at 37 °C^35,36,37. Cells were allowed to migrate towards fMLP at 37 °C for 1 h. The number of migrating cells in the lower chamber was counted using an automated cell counter (Invitrogen, Waltham, MA, USA).