Neutrophil and Monocyte Migration Assay

The migration of unstimulated neutrophils or monocyte and chemotaxis of cells were measured as previously reported (5 (link)). Briefly, advanced RPMI (Thermo Fisher, Cat#12633–012) with the chemoattractants CXCL1 (BioLegend Cat#573702), CCL2 (R&D Systems, Cat#479-JE), or N-Formylmethionyl-leucyl-phenylalanine (fMLP; Sigma-Aldrich Cat#F3506) was placed in the bottom of the 3-μm-pore Transwell system (Neuro Probe Cat#101–3). Neutrophils were incubated with PSB12379 (Tocris) or 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) (Sigma-Aldrich, Cat#C101) in advanced RPMI for 10 min before being plated on top of the Transwell (0.1×10⁶ cells) and were incubated at 37°C, 5% CO₂ for 1 h. 10μl medium was taken from the bottom wells and cells were counted by a hemocytometer. The quantification of migrated neutrophils was done with the formula N=n×10⁴×0.029 (reflective of 29μl medium in the bottom well).