Reactive oxygen species (ROS) formation

The ROS fluorescent indicator dihydrorhodamine 123 (DHR123, Molecular Probes) was used to monitor ROS levels during cell activation, and the procedure was modified as described [24]. Briefly, freshly isolated neutrophils (1 × 10⁶ cells/ml) were incubated in 1 μM DHR123 in Ca²⁺-free HBSS at 37 °C for 20 min. After washing with Ca²⁺-free HBSS, the DHR123-loaded cells were resuspended in Ca²⁺-containing HBSS and treated with 100 nM fMLP for 15 min at 37 °C. To stop the reaction, a 5-fold volume of ice-cold Ca²⁺-containing HBSS was added and kept at 4 °C in the dark. The aggregated cells were removed by filtration through a 35-μm cell strainer snap cap on a flow cytometry tube (Falcon, Corning, Cambridge, MA). Then, neutrophils were recognized via a BD Accuri C6 Flow Cytometer (BD Biosciences, San Jose, CA, USA) on the basis of forward light scatter (FSC) and side light scatter (SSC), which identified neutrophils and excluded other cell types (a few contaminating lymphocytes and red blood cells), dead cells and debris from the analysis. Appropriate gates were set to take the changes in the light-scattering pattern of fMLP-activated neutrophils into consideration during analysis. FL1 channels (green fluorescence) were chosen to detect the fluorescence of rhodamine 123, which was oxidized from DHR123 by ROS.