Cells were cultured in 6-well plates with a density of 5×105 cells per well and incubated for 12 hours. After applying various treatments, the cells underwent the following procedure to assess intracellular oxidation levels.
For intracellular GSH detection, MNNG/HOS cells were suspended in 200 µL of PBS per well and disrupted using ultrasonication. The disrupted cells were mixed with GSH probe precipitant in a 1:1 ratio (v/v) and then centrifuged at 3500 rpm for 10 minutes. The supernatant’s absorbance at 405 nm was measured, and GSH levels were determined using a GSH assay kit as per its instructions. Intracellular lipid peroxidation (LPO) was assessed by measuring MDA levels using an LPO MDA assay kit (S0131S, Beyotime, Shanghai, China) following established methods.32 (link) For visualizing cellular ROS, the fluorescent probe 2’,7’-dichlorodihydrofluorescein diacetate (DCFH-DA) was employed. DCFH-DA emits green fluorescence upon reacting with ROS. The treated cells were rinsed, stained with DCFH-DA (10 µM) for 20 minutes, and then washed with PBS twice. Subsequently, the cells were observed and imaged using a fluorescence microscope.
For intracellular GSH detection, MNNG/HOS cells were suspended in 200 µL of PBS per well and disrupted using ultrasonication. The disrupted cells were mixed with GSH probe precipitant in a 1:1 ratio (v/v) and then centrifuged at 3500 rpm for 10 minutes. The supernatant’s absorbance at 405 nm was measured, and GSH levels were determined using a GSH assay kit as per its instructions. Intracellular lipid peroxidation (LPO) was assessed by measuring MDA levels using an LPO MDA assay kit (S0131S, Beyotime, Shanghai, China) following established methods.32 (link) For visualizing cellular ROS, the fluorescent probe 2’,7’-dichlorodihydrofluorescein diacetate (DCFH-DA) was employed. DCFH-DA emits green fluorescence upon reacting with ROS. The treated cells were rinsed, stained with DCFH-DA (10 µM) for 20 minutes, and then washed with PBS twice. Subsequently, the cells were observed and imaged using a fluorescence microscope.
