96 well microtiter plate reader

The cell viability assay was carried out as described [46 (link)] using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Merck) with minor modifications. BAECs were seeded at a density of 1.0 × 10⁴ per well in a 96-well culture plate (four replicates for each treatment), and incubated with ZEN at various concentrations (0, 10, 30 or 60 μM) for 24 h or with 30 μM ZEN for various time points (4, 8, 16 or 24 h). After the ZEN treatments, the cells were incubated with 5 mg/mL MTT and further incubated for 1 h at 37 °C. The cells were then treated with dimethylsulfoxide (DMSO) for 10 min, and the absorbance was read at 570 nm using a 96-well microtiter plate reader (BioTek Instruments, Winooski, VT, USA).

Intracellular ROS was measured using the oxidation-sensitive fluorescent probe DCF-DA (Merck), in an assay based on the ROS-dependent oxidation of DCF-DA to 2′,7′-dichlorofluorescein (DCF), as described previously [47 (link)] with minor modifications. Briefly, BAECs grown in 96-well culture plates were incubated with 20 μM DCF-DA for 30 min. The DCF-DA solution was removed and the cells were washed with DPBS. The cells were then incubated with NAC for 3 h before ZEN treatment and incubated for an additional 24 h at 37 °C under 5% CO₂ after the addition of ZEN. The deposited intracellular DCF-DA was measured using a 96-well microtiter plate reader (ex 485 nm/em 530 nm; BioTek Instrument).

The cell proliferation of the compounds was tested using the MTT method, as reported previously [12 (link)], with 1‰ DMSO medium as the blank control. Human neuroblastoma cell line SK-N-BE (2) was maintained from American type culture collection (ATCC) and prepared in our lab. Compounds 1–3 were dissolved in DMSO as reserve liquid (0.1 M), which were diluted into different concentrations (2.5, 5, and 10 μM) with cell culture Medium (Eagle’s Minimum Essential Medium). SK-N-BE (2) cells (1.0 × 10⁴ cells/mL, 90 μL/well) were seeded on the 96-well plates and incubated overnight, then treated with each test compound at various concentrations (10 μL/well) at 37 °C in a humidified atmosphere containing 5% CO₂ for 72 h. After the incubation, MTT was added to each well as described [12 (link)]. Optical density at 595 nm was measured in a 96-well microtiter plate reader (BioTek, Winooski, Vermont, USA). The optical density of formazan formed in control (untreated) cells was taken as 100% of viability. Three replicate wells were used for each control and test concentrations per microplate, and the experiment was repeated three times.