3.7. Mitochondrial Membrane Potential (ΔΨm) Measurement

JC-1 is a cationic dye that accumulates in mitochondria in a membrane potential-dependent manner. At low membrane potentials, JC-1 exists in monomeric form and emits green fluorescent (490 nm excitation/530 nm emission), whereas at high membrane potential is in an aggregated form and exhibits a red fluorescent (530 nm excitation/590 nm emission). A decrease in membrane potential is, therefore, indicated by a fluorescence emission shift from green to red. A 1.5 mM stock solution in DMSO was prepared according to the manufacturer’s instructions. 72-hpf zebrafish embryos were incubated in the dark for 30 min with 5.0 μM JC-1 solution in PBS. After incubation time, embryos were washed three times with E3 medium and then treated with selected concentrations of ZnO NPs, with or without resveratrol. Fluorescence intensity was measured with GENios plus microplate reader (Tecan, Männedorf, CH) with both red (535–590 nm) and green (485–535 nm) channels, using a 96-multiwell dark plate with 5/6 embryos per well. The images were captured using a fluorescent microscope with red and green channels. Both qualitative and qualitative analyses were performed in vivo with tricaine-anesthetized embryos. Data (representative of 5 independent experiments with a total number of 50/60 embryos for each experiment) are expressed as a means ± SD of the relative fluorescence unit (RFU) values and have been normalized to background fluorescence (wells containing only solution and wells containing zebrafish embryos untreated with JC-1 probe). The results expressed as the ratio of average red/green fluorescence signals represent quantification of the degree of Δψm level [43,44,45].