2.11. Determination of reactive oxygen species (ROS)

Dichloro-dihydro-fluorescein diacetate (DCFH-DA), purchased from Merck, was used as fluorescence probes. After incubation, MH-S cells were rinsed with PBS buffer twice and 10 μM DCF-DA probes were added before incubating at 37 °C with 5% CO₂ for 20 min. After incubation, the fluorescence probes were removed, and MH-S cells were washed with PBS buffer twice for fluorometric analysis. Quantification of ROS was measured using a fluorescence microplate reader (Varioskan LUX Multimode Microplate Reader, Thermo) with an excitation wavelength of 488 nm and an emission wavelength of 525 nm. The ROS level in each well was normalized with the control group (100%) and corrected by dividing it with the cell viability [31]. The generation of ROS in cells was observed and captured using a fluorescence microscope (Carl Zeiss Inc., Oberkochen, Germany).