Respiratory Burst Activity
Generation of ROS was evaluated by flow cytometry using the technique of Smith and Wiedemann22. Cells were incubated with DHR 123 (50 μL) and PBS (450 μL) at 37°C for 10 minutes. Cells were stimulated with 1 μL (16 μM) phorbol 12-myrisate 13-acetate (PMA) for 20 minutes at 37°C. The reaction was then halted by placing samples on ice. Fluorescence intensity was assessed by flow cytometry and expressed as mean fluorescent intensity (MFI). DHR 123 has been shown to detect intracellular H2O2 and OH radical production22.