Intracellular ROS Measurement in RAW264.7 Cells

The level of intracellular ROS was determined by a change in fluorescence resulting from the oxidation of the fluorescent probe, DHR123. Briefly, 5 × 10⁵ RAW264.7 cells were exposed to KF for 30 min. After incubation, cells were then incubated with SNP (0.25 mM), an inducer of ROS production, at 37°C for 2 h. Cells were incubated with 20 μM of the fluorescent probe DHR123 for 1 h at 37°C. The degree of fluorescence, corresponding to intracellular ROS, was determined using a FACScan flow cytometer (Becton-Dickinson, San Jose, CA), as reported previously [3 (link), 33 (link), 35 (link)]. Briefly, the RAW264.7 cells treated with KF, SNP, and DHR123 were washed with a staining buffer (containing 2% rabbit serum and 1% sodium azide in PBS) and incubated for a further 45 min on ice. After washing three times with staining buffer, stained cells were analyzed on a FACScan flow cytometer (Becton-Dickinson, San Jose, CA, USA).