Reduced glutathione gsh assay kit

The GSH/GSSG ratio, GSH levels and cysteine levels were detected by Reduced glutathione (GSH) assay kit (Nanjing jiancheng, China), Total glutathione / Oxidized glutathione assay kit (Nanjing jiancheng, China) and Cysteine content test kit (Nanjing jiancheng, China) on the basis of the manufacturer’s instructions. The transfected cells were lysed in culture dishes containing a lysis buffer, and 0.5 ml supernatant was taken from the resulting lysates which were centrifuged. Two ml of the application solution was added and mixed evenly, and then centrifuged at 3000 g for 10 min. Lastly 1 ml of the supernatant was taken for color reaction. The measurements were conducted with a UV–visible spectrophotometer.
A detailed description of the materials and methods used in this study can be found in the online supplementary material.

Antimycotics (#15240062), fetal bovine serum (FBS, #10091148), RPMI 1640 medium (#A1049101), and Dulbecco's modified Eagle's high glucose medium (#11965118) were purchased from HyClone (HyClone, Logan, UT, USA). Cell Counting Kit-8 (CCK-8, #CK04) was obtained from Dojindo (Dojindo Laboratories, Kumamoto, Japan). The malondialdehyde (MDA) assay kit (#A003-1-2) and reduced glutathione (GSH) assay kit (#A006-2-1) were purchased from Jiancheng (Jiancheng, Nanjing, China). The DAPI staining solution kit (#MA0127) and zinc protoporphyrin-9 (#MB4231) were purchased from Meilui (Meilui, Dalian, China). Liperfluo (#L248) was purchased from Dojindo (Dojindo Laboratories, Shanghai, China). 2′,7′-Dichlorodihydrofluorescein diacetate (DCFH-DA, #S0033S), Annexin V (#C1062M-1), and Propidium Iodide (PI, #C1062M-3) were purchased from Beyotime (Beyotime, Shanghai, China). For western blot analysis, antibodies against HO-1 (#ab13243), GPX4 (#ab40993), Nrf2 (#ab137550), and β-actin (#ab8226) were purchased from Abcam (Abcam, Cambridge, MA). The Iron Assay Kit (#MAK025), dimethyl sulfoxide (DMSO, #D2650), curcumin (#C1386), z-VAD-fmk (#V116), ferrostatin-1 (#SML0583), and deferoxamine (#D9533) were purchased from Sigma (Sigma, St Louis, USA). These compounds were dissolved in DMSO (at final concentrations less than 0.1% (v/v)).

The levels of total antioxidant capacity (T-AOC), inhibition ability of anti-superoxide anion free radicals (ASAFR), reduced glutathione (GSH) and H₂O₂ in the plasma were measured with the total antioxidant capacity assay kit, the inhibition and product superoxide anion assay kit, the reduced glutathione (GSH) assay kit and the hydrogen peroxide assay kit respectively, according to the instructions (Jiancheng Bioengineering Institute, Nanjing, China). The plasma of silkworms was collected according to the method described above, and incubated with corresponding reagents for 5–10 min. The multi-functional enzyme marker was used to measure the OD value of T-AOC, GSH and H₂O₂ at 405 nm, and the OD values of the content of the inhibition and production of superoxide anion radical at 550 nm.