3.6. ROS Measurement

ROS levels were determined by using the ROS fluorescent probe Carboxy-H2DCFDA. The non-fluorescent probe molecule diffuses into cells where once deacetylated by cellular esterase, is converted into a highly fluorescent green inform presence in the presence of ROS. Zebrafish embryos (72 hpf) were first incubated with 5.0 μM of the probe for 60 min, in the dark, and then washed three times with E3 medium before exposure to the selected concentrations of ZnO NPs dispersion, with or without 5 µM resveratrol. Before proceeding to further analysis (imaging and quantitative assessment), embryos were anesthetized with tricaine. Fluorescence intensity was measured with GENios plus microplate reader (Tecan, Männedorf, CH) at the excitation wavelength of 485 nm and an emission wavelength of 535 nm, using a 96-multiwell dark plate with 5/6 embryos per well. Results were normalized to background fluorescence, in both wells containing only solution and wells containing zebrafish embryos untreated with ROS probe. Data are representative of five independent experiments with a total number of 50/60 embryos for each experiment and were expressed as a means ± SD of the relative fluorescence unit (RFU) values. Imaging and qualitative ROS evaluation were carried out by observation of live embryos under a fluorescence microscope Zeiss (SteREO Discovery V8 Microscope) [65,66,67,68,69].