Gsh gssg assay kit

The concentrations of total glutathione (T-GSH) were examined with a GSH/GSSG assay kit (Beyotime Institute of Biotechnology, China) based on an enzymatic method according to the manufacturer’s instructions. A total of 30-40 oocytes from each group were mixed with 30 μl of protein scavenger M solution supplied by the kit and vortexed thoroughly for 5 min, then the mixture was frozen at -80°C for 2 min and thawed at 38.5°C repeatedly for three times. The mixture was centrifuged at 10,000 rpm for 10 min at 4°C and put on ice for 5 min. The absorbance was monitored continuously at 412 nm with a microplate spectrophotometer (PerkinElmer, USA) for 25 min, with a reading recorded every 5 min and regarded the readings at the time point 25 min as the final absorbance values.

HCT116 cells were seeded into 6-well plates, and cultured for 24 h in the presence and absence of 5-Fu for 48 h. Cells were harvested and NADPH/NADP⁺ and GSH/GSSG ratios were determined using an NADPH/NADP⁺ Assay Kit (S0179, Beyotime) and GSH/GSSG Assay Kit (S0053, Beyotime), respectively, according to the manufacturer’s instructions.

Adenosine triphosphate (ATP) and GSH levels in SHG140 and U87 cells were measured using enhanced ATP assay kit (Beyotime, S0027, China) and GSH/GSSG assay kit (Beyotime, S0053, China). Each experiment had been repeated three times.

The contents of total glutathione (T-GSH) were examined through an enzymatic method by using a GSH/GSSG assay kit (Beyotime, Shanghai, China) according to the manufacturer’s instructions. A total of 50 oocytes from each group were placed into a small conical tube containing 30 µL of protein scavenger M solution supplied with the kit. Afterward, tube contents were vortexed thoroughly for 5 min, then the mixture was frozen at liquid nitrogen for 2 min and thawed in a water bath at 37 °C repeatedly for 3 times. Subsequently, mixture was centrifuged at 10,000 rpm for 10 min at 4°C and placed on ice for 5 min using a 96-well plate. The samples or standard in the sequence were added and mixed accordingly. Immediately, absorbance was observed at 405 nm with a microplate reader, for 25 min, with a reading recorded for every 5 min. A standard curve was developed for the determination of the GSH content of each sample. The GSH concentration was calculated by dividing the total concentration of each sample by the total number of oocytes present in the sample (pmol/oocyte).

The MDA concentration of tissue and cell lysates were assessed using the MDA Assay Kit (S0131, Beyotime, Shanghai, China) according to the manufacturer’s instructions.
Total GSH and GSSG levels of tissue and cell lysates were measured using the GSH-GSSG Assay Kit (S0053, Beyotime, Shanghai, China) according to the manufacturer’s instructions. A standard curve was established by the addition of GSH to PBS, and net GSH concentration was determined as subtract GSSG concentration from total GSH content.

The level of GSH/GSSH was detected using the GSH/GSSG assay kit (Beyotime, Shanghai, China). The detection principle was that GSH reacted with the chromogenic substrate DTNB to produce yellow TNB. The amount of total cellular GSH can be calculated by detecting the absorbance at 412 nm with a microplate reader. After Ce6, Fe-CDs, Fe-CDs@Ce6, and Fe-CDs@Ce6 + PDT treatments, B16 cells were washed with PBS, recentrifuged at 1000g for 3 min, and the supernatant was aspirated. The cells were lysed by two freeze–thaw cycles in liquid nitrogen and 37 °C water bath. The molar concentrations of GSH and GSSG were calculated according to the standard curve, and the GSH contents were determined as the protein content per unit weight.
The generation of singlet oxygen (¹O₂) in vitro was detected with a singlet oxygen detection kit (S36002, Thermo fisher, USA). Briefly, 25 µL of 50 µM SOSG was added into the solutions containing free Fe-CDs (200 µg/mL), Ce6 (80 µg/mL), and Fe-CDs@Ce6 (200 µg/mL), followed by 660 nm laser irradiation at 100 mW/cm² for different time periods (0, 10, 20, 30, 40, 50, 60 min). Subsequently, oxidized SOSG was detected by a microplate reader to represent ¹O₂ production. The blank aqueous solution was used as a negative control.