4.8. Determination of ROS Levels

The generation of intracellular oxidative stress was determined using the dihydroethidium (DHE) probe as previously described [58]. In brief, A549 cells were grown in complete media, seeded in 12-well plates (50.000 cells/mL) allowed overnight to attach. Cells were exposed for 24 h to DMSO (Control) or 0.1 and 0.5 µg/mL of crude poison secretion (L) or A, B, and C fractions. Then, culture media was replaced by a solution containing 5 µM DHE in Hank’s balanced salt solution (HBSS) and incubated for 20 min in the dark. After this time, cells were washed, trypsinized, and resuspended in 200 µL of HBSS and measured by FACSCanto flow cytometer.