Spectramax i3x

The intracellular ROS levels were measured using a Reactive Oxygen Species Assay Kit (Beyotime, Shanghai, China) according to the manufacturer’s instructions. Briefly, the cells were seeded into 96-well plates and exposed to various concentrations of T-2 toxin for 24 h. Following treatment, the cells were incubated with DCFH-DA for 1 h at 37 °C and measured using a microplate reader (Spectra Max i3x, Molecular Devices) with 488 nm excitation and 525 nm emission.
Dihydroethidium (DHE, Beyotime, Shanghai, China)) is used to detect the level of superoxide anion in cells. The cells were seeded into 12-well plates and exposed to various concentrations of T-2 toxin for 24 h. Following the treatment, the cells were incubated with DHE for 1 h at 37 °C and measured using a Microscope (Spectra Max i3x, Molecular Devices, Sunnyvale, CA, USA) with 488 nm excitation and 525 nm emission.

Cell viability was assessed using an MTS assay (G3582, Promega) at 24 h. HepG2 cells were seeded in a clear-bottom 96-well plate at 10,000 cells per well and stimulated, as described before. The MTS reagent was added and incubated at 37°C for 1 h before detection of absorbance at 490 nm on a plate reader (SpectraMax i3x, Molecular Devices). Oil O Red was used to assess intracellular fat accumulation within cells plated at 300,000 cells per well in 6-well plates. Cells were stimulated as previously described, washed with PBS, and fixed with 4% paraformaldehyde for 30 min. The Oil O Red stock solution was prepared as 0.5% Oil O Red (NC0961554, Thermo Fisher Scientific) in isopropanol and diluted to 60% in nanopure water fresh for each use. The working Oil O Red solution was filtered before staining for 10 min at room temperature. After staining, cells were washed three times with PBS and then imaged (EVOS Core XL, Thermo Fisher Scientific). To elute dye for quantification, 250 µl of 100% isopropanol was added to stained cells. Cells were then rocked at room temperature for 10 min in isopropanol before transferring 75 µl of the isopropanol solution from each well to a 96-well plate. The absorbance of eluted dye was measured at 540 nm on a plate reader (SpectraMax i3x, Molecular Devices). Statistics were carried out on Prism 9.1 (Graphpad).

At the end of the treatment period, cells seeded in the 96-well plate were washed with PBS and supplemented with a mixture consisting of 25 μL of caspase reagent (Promega, USA) and 25 μL of fresh medium. The reagents were gently mixed on a plate shaker at 250 rpm for 1 min and incubated at room temperature for 30 min. Following incubation, luminescence was measured using a plate reader (SpectraMax i3x; Molecular Devices, USA). Next, 50 μL of 2× concentrated CyQUANT reagent (Thermo Fisher Scientific, New Zealand) was added to each well. The plate was then incubated at room temperature for 10 min in the dark, and fluorescence was measured using the plate reader (excitation at 480 nm and emission at 520 nm, SpectraMax i3x; Molecular Devices, USA). Caspase activity (relative luminescent units) was then normalized to the cell numbers measured using the CyQUANT reagent (relative fluorescence units) and represented as caspase activity/cell number.

The ability of O. sinensis melanin to scavenge free radicals was measured using the DPPH method, and the procedure was adapted from Surendirakumar et al. [23 (link)]. Briefly, different concentrations of O. sinensis melanin were prepared with DMSO, and 0.05 mg/mL DPPH (ethanol solution) was mixed at a volume ratio of 1:1. After incubation in the dark at 24 °C for 30 min, a spectrophotometer (SpectraMax i3x, Molecular Devices, Shanghai, China) was used to read the absorbance at 517 nm. Trolox was used as a control. The antioxidant activity of O. sinensis melanin was also examined using the ABTS method as described by Khemakhem et al. [41 (link)]. Approximately 7.4 mM ABTS storage solution and 2.6 mM K₂S₂O₈ storage solution were prepared, mixed with equal volumes, and reacted at 4 °C for 12–15 h (protected from light), and then diluted with ethanol to OD734 = 0.7 to obtain the ABTS working solution. The samples and ABTS working solution were mixed at a volume ratio of 1:4, kept in the dark at 24 °C for 30 min to react, and a spectrophotometer (SpectraMax i3x, Molecular Devices, Shanghai, China) was used to read the absorbance at 714 nm.
The ability to scavenge the DPPH (or ABTS) radical was calculated using the following equation:
where A₀: DPPH (or ABTS)+ DMSO; A₁: DPPH (or ABTS)+ melanin; and A₂: melanin + ethanol.

The products were dissolved in deionized water at the concentration of 1 mg/mL. The absorption spectra were measured using a UV-visible spectro-photometer (Molecular devices lnc, San Jose, CA, USA, spectraMax i3x) to determine the absorption peaks. The emission spectra were recorded with an excitation wavelength of 488 nm using a fluorescent photometer (Molecular devices lnc, spectraMax i3x). For the fluorescence dot assay, 50 μL of a different 1 mg/mL solution was added onto the transparent glass, and the photo was taken in a different channel.