Molecular probes dcfh da

The production of ROS in zebrafish larvae was determined using membrane-permeable fluorescent dye 2’,7’-dichlorodihydrofluorescin diacetate (Molecular Probes DCFH-DA; Thermo Fisher Scientific, USA) according to the method of Zeng et al. [23 (link)] with a minor modification. Three replicates of twenty larvae that had been treated with 0.3, 1.3, 5, 20 and 80 ppb EZA for 3 days were homogenized in CytoBuster protein extraction buffer (Merck, Germany). And the homogenate was centrifuged at 15,000×g at 4 °C for 20 min. The supernatant was incubated for 30 min with DCFH-DA in PBS (pH7.4). The fluorescence intensity was measured using a microplate reader (SpectraMax M5, Molecular Devices) with excitation and emission at 485 nm and 530 nm, respectively. The ROS levels were estimated based on a DCF (dichlorofuorescein) standard curve, and expressed as an arbitrary unit (μg DCF /mg protein) and as a percentage of a control.

The average density of intracellular ROS was evaluated in cells loaded with the redox-sensitive dye, dichloro-dihydro-fluorescein diacetate (Molecular Probes DCFH-DA; Thermo Fisher Scientific, Inc.). SH-SY5Y cells, with or without lycopene pretreatment, were seeded into 96-well culture plates at a density of 4×10⁴ cells/well, exposed for 24 h to 400 µmol/l H₂O₂, washed twice with PBS, stained with 20 µmol/l DCFH-DA in the dark for 30 min and harvested. The cells were then dissolved with 1% Triton X-100 (Sigma-Aldrich). Fluorescence was observed under a fluorescent light microscope, and measured at excitation and emission wavelengths of 485 and 530 nm, respectively using a fluorescence spectrometer (HTS 7000; PerkinElmer, Inc., Waltham, MA, USA). The ROS levels were expressed as arbitrary unit/mg protein and as a percentage of the control.