2.5. Determination of intracellular reactive oxygen species (ROS)

Production of ROS in the living cells was measured using cell-permeable ROS detecting fluorescent dye CM-H2DCFDA. Following treatment procedures, cells were gently harvested with warm Hank’s Balanced Salt Solution (HBSS, 1X, Cellgro), transferred from flasks into tubes and spun. Pellets were suspended in HBSS and incubated with 10 μM of CM-H2DCFDA at 37°C for 30 minutes. After short centrifugation the excess dye was aspirated; cells were suspended with warm HBSS and transferred into 24-well plates. The end-point arbitrary fluorescent units were recorded on an SLM 8000 Thermospectronic fluorometer, setting excitation and emission wavelengths at 485 nm and 538 nm, respectively.

For in situ measurements, cells were grown in 6-well plates with coverslips inserted on them and processed for total intracellular ROS assay. Fluorescent images were captured with an Olympus BH-2 microscope at 200x magnification.