Measuring Neutrophil Oxidative Stress

Dihydrorhodamine 123 (DHR 123; Invitrogen, Waltham, MA) was used to detect ROS production. Neutrophils 10⁶/ml were seeded in a 96-well plate in 100 μl DMEM (DMEM (Gibco, USA) or placed in an Eppendorf tube in 500 μl DMEM. Then, DHR123 (final concentration 5 μM) was added for 10 minutes and cells were washed with PBS. Neutrophils were incubated with 100 µl or 500 µl of the stimulating factors for 1 h at 37°C, 5% CO₂. Cells stimulated by PMA (25 nM) were used as the positive control. The fluorescence of oxidized rhodamine 123 was measured using a microplate reader (H1, Biotek), or flow cytometry (BD Fortessa).