Oxidative Burst Test

Neutrophil’s ROS production was quantified by the burst test kit (Orpegen Pharma Heidelberg Germany). In brief, 100 µl of heparinized peripheral whole blood was incubated with 20 µl (2 × 10⁷ cells) of either unlabeled opsonized E. coli as particulate stimuli or wash buffer as a control. Blood samples were treated with 20 µl (0.32 mM) of either phorbol 12-myristate 13-acetate (PMA: 200× stock solution, 1.62 mM, 5 µl of stock solution was diluted in 1 ml of wash buffer and then 20 µl was used) or 20 µl (0.2 mM) of chemotactic synthetic peptide N-formyl-MetLuePhe (fMLP: 200× stock solution, 1 mM) to assess high and low oxidative burst capacity, respectively. Cells were incubated for 20 min at 37° in water bath, 20 µl of substrate (dihydrohodamine-123) was then added, and samples were further incubated for 20 min. RBCs were lysed, and after two washes with wash buffer, cells were stained for neutrophil markers mentioned above. After washing with PBS, cells were analyzed by flow cytometry. Oxidative burst was quantified according to the percentage of CD11b+ CD16+ neutrophils producing ROS. The production of ROS was measured by flow cytometry using the oxidation of dihydrohodamine-123 to rhodamine which emits green fluorescence.