Chemotaxis Assay of Neutrophils

Chemotaxis assay was performed on freshly isolated WT and Nrf2 KO PMNs using transwell migration assay, as previously described [37 (link)]. Briefly, PMNs (1 × 10⁶ cells) were added to the upper chamber of Transwell filters (3 μm pore diameter, Costar). These chambers were placed in 24-well cell culture plates containing 600 μL assay buffer without chemoattractant or with N-Formylmethionyl-leucyl-phenylalanine (fMLP, 1 μM) (Sigma-Aldrich), increasing concentrations of CXCL2 (0.5–200 nM) and increasing concentrations of CXCL12 (50–400 nM) (both from BioLegend). In some experiments, cells were preincubated with AMD3100 octahydrochloride 50 nM (Sigma-Aldrich) for 30 min before being placed in the upper Transwell chamber to confirm the specificity of CXCR4-dependent migration. Chambers were then incubated for 60 min at 37°C with 5% CO2 and the cells that had migrated to the bottom chamber were recovered and stained with antibodies against Ly6G (PercP. Cy5.5) and CD11b (FITC) (both from BioLegend) for flow cytometry analysis (FACS Calibur). Chemotactic indexes were then calculated by dividing the number of PMNs counted in chemokine-stimulated wells by the number of PMNs counted in filter-free wells (input well without any chemokine).