Gsh colorimetric assay kit

The GSH Colorimetric Assay Kit (BioVision) was used to measure the caspase activities according to the manufacturer's instructions. K562 cells were untreated or treated with the indicated doses of MG132 for 24 h. The cells were then incubated with glutathione reductase and the GSH substrate for 5 min. The absorbance values were obtained with a microplate reader (Bio-Rad) at a wavelength of 405 nm.

We measured lipid peroxidation, glutathione (GSH) concentration, and superoxide dismutase (SOD) activity as oxidative stress markers on day 3 after injury. Snap-frozen cortex was homogenized with ultrasonic waves in a bead homogenizer. Cortical lipid peroxide content was determined using the modified thiobarbituric acid (TBA)-reactive substances assay method as described previously [21 (link)]. Briefly, an aliquot of the homogenate was added to an equal volume of 0.67% (w/v) TBA. Subsequently, 800 μL of the supernatant were mixed with 100 μL of 0.1 M TBA and heated at 100°C for 10 minutes. After cooling, the absorbance was read at 532 nm using a SpectraMax 250 microplate reader (Molecular Devices, Sunnyvale, CA, USA). A malondialdehyde (MDA) standard was prepared from 1,1,3,3-tetraethoxypropane. GSH levels and SOD activity were determined using the GSH colorimetric assay kit (BioVision, Milpitas, CA, USA) and SOD assay kit (Cell Biolabs, CA, USA), respectively, according to the manufacturer’s instructions.

The DCFDA cellular reactive oxygen species (ROS) assay was performed following the manufacturer's instructions (ab113851, Abcam, UK). Briefly, cells were incubated with DCFDA (20 μM, 100 μL/well) for 0.5 h at 37 °C in the dark. DCFDA is a fluorescent probe that can detect ROS. After diffusion into cells, non-fluorescent DCFDA is deacetylated by cellular esterases and then oxidised by ROS into the fluorescein 2′,7’ - dichlorofluorescein (DCF), which cannot diffuse out of cells. As DCF is fluorescent, it can be detected using fluorescence spectroscopy, with excitation and emission wavelengths of 495 nm and 529 nm, respectively. The level of lipid ROS in cells or tissues was evaluated using flow cytometry after 20 min of staining with 1.5 mM C11-BODIPY (Invitrogen, Carlsbad, CA, USA) [2 (link)]. In cell lysates subjected to different treatments, cellular glutathione (GSH) levels were determined using a GSH colorimetric assay kit (BioVision Inc., Milpitas, CA, USA) following the manufacturer's instructions.