Neutrophil NADPH oxidase and apoptosis assays

Single cells suspensions were obtained one day p.i. from mouse ears as described above. Cells were surface stained with Brilliant Violet 711 anti-Ly6G, Brilliant Violet 421 anti-CD45 clone 30-F11, and APC-Cy7 anti-CD11b clone M1/70 from Biolegend. To assess the ability of neutrophils to activate the phagocyte NADPH oxidase, cells were incubated in PBS with 100 ng/mL of PMA (Sigma Aldrich) and 10 μM dihydrorhodamine 123 (DHR123, Sigma Aldrich) for 15 minutes at 37°C, 5% CO₂, and then washed in PBS and analyzed by flow cytometry. To assay for apoptosis, cell suspensions were resuspended in 100 μL Annexin V binding buffer (Biolegend) and incubated with 5 μL of APC Annexin V (Biolegend) for 10–15 min at room temperature in the dark. Within 10 minutes prior to analysis by flow cytometry, 10 μL of Propidium Iodide Staining Solution (Biolegend) was added to each sample.