Reduced gsh assay kit

Rats at 7 dpi were anesthetized and perfused with pre-cooling sterile saline for removal of erythrocytes in spinal cords. The injured tissue was moved to a sterile glass dish. Spinal dura mater and nerve roots were carefully peeled off. The tissues were homogenized in 9-fold (volume/weight) of normal saline. After centrifugation at 600 × g for 10 minutes, the supernatant was collected and assayed with a tissue iron assay kit (A039-2-1) and reduced-GSH assay kit (A006-2-1; Nanjing Jiancheng Bioengineering Institute, Nanjing, China). The optical density (OD) value was measured at 520 nm (for tissue iron) and 405 nm (for GSH). Iron and GSH concentrations were calculated following the manufacturer’s instructions.

The concentration of intracellular GSH was measured using a reduced GSH assay kit (Nanjing Jiancheng Bioengineering Institute, China). Briefly, 4T1 cells were seeded in 12-well plates at a density of 1×10⁵ cells/well, and wells were treated individually with PBS, GA, Ce6, GA&Ce6 and HA-GA@Ce6 for 4 h (Ce6 = 2.5 µM, GA = 1.5 µM). Untreated cells were used as negative controls. N-Ethylmaleimide (NEM), a GSH inhibitor, was used as a positive control. During disruption of intracellular redox homeostasis, ROS concentrations should increase. The cells were then collected by trypsinization and processed according to the assay kit instructions.

Glutathione (GSH) levels were quantified using a commercial reduced GSH assay kit (Jiancheng), and the amount of GSH was expressed in terms of micromoles of GSH per gram of proteins. Glutamate cysteine ligase (GCL), also known as γ-glutamylcysteine synthetase, is the first rate-limiting enzyme in GSH synthesis, and has both catalytic (GCLC) and modifier (GCLM) subunits. Total GCLM and GCLC levels were quantified by real-time polymerase chain reaction (PCR) according to the methods mentioned in the following sections.

The cells were digested using trypsin; after washing with PBS twice, the cells were added to 300 μL of PBS and subsequently crushed using an ultrasonic cell crusher at 300 W. This process was repeated four times, with a 30 s interval for each 5 s. Animal tissues were mixed with PBS at a ratio of 1 mL per 0.1 g and then ground using a tissue grinder. The tissues were then crushed four times with an ultrasonic cell crusher at 300 W, with a 30 s interval for each 5 s. Hippocampal tissue was processed in the same steps as above after grinding. Finally, the levels of MDA and reduced GSH in the serum, hippocampus and cells were measured according to the manufacturer's instructions for the MDA assay kit (Beyotime, China) and the reduced GSH assay kit (Nanjing Jiancheng Biotech, China), respectively.