Facs calibur

In some experiments, sulforaphane (SFN) (Sigma-Aldrich, St. Louis, MO, USA) was used in order to strongly activate Nrf2 in WT PMNs. Cells (1 × 10⁶/ml) were pre-incubated with SFN 1 μM for 4 h at 37 °C with 5% CO₂. As SFN can be toxic in some conditions, we checked that none of the tested SFN concentrations (1 and 5 μM) were either toxic or induced apoptosis within 4 h, using the Annexin V/7-amino-actinomycin (AnnV/7AAD) counterstaining (BioLegend) followed by flow cytometry (FACS Calibur). Cells positive for Annexin V and negative for 7-AAD were considered as apoptotic cells (consistently under 3%), whereas double positive cells were considered as necrotic cells (consistently under 8%) (S2 Fig).

For detection of apoptotic and/or necrosis in treated tumor cells using flow cytometry, cells were stained with FITC Annexin V Apoptosis Detection Kit with 7-AAD (BioLegend, San Diego, CA, USA). Stained cells were analyzed using a fluorescence-activated cell sorting (FACS) Calibur (BioLegend, San Diego, CA, USA).

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).