In vitro chemotaxis assay

The in vitro chemotaxis assay was performed using a transwell insert plate with an 8 μm pore size (SPL). 10 μg of wild-type Gcf and 100 μg of mAbs (5H6 or 3A5) were mixed for 30 min at 4°C, and the mixture was added to the lower chamber of the insert plate. As a negative control, serum-free media alone or serum-free media that contained antibody (5H6 or 3A5) was added to the lower chamber. As a positive control, serum-free media that contained 10 μg of wild-type Gcf or media with 10% FBS was added to the lower chamber. 5 × 10⁵ THP-1 cells (ATCC, Manassas, VA) expressing CX3CR1 receptor were added to the upper chamber of the plate. The assembled plates were incubated for 5 hr at 37°C. Cells migrated to the lower chamber were counted, and the percent migration was determined using the following formula: 100 × {[(average number of migrated cells/area of the microscope viewing field) × area of the transwell insert]/number of cells seeded}.