Glutathione reductase assay kit

Resveratrol was purchased from Aladdin (Shanghai, China). NAC (N-acetyl-L-cysteine) and Dimethyl sulfoxid (DMSO) were purchased from Sigma (St. Louis, MO, USA). The MDA assay kit, SOD activity assay kit, antioxidant capacity assay kit, glutathione peroxidase assay kit and glutathione reductase assay kit were obtained from Nanjing Jiancheng Bioengineering Institute (Nangjing, China). Dulbecco’s modified eagle medium (DMEM) and fetal bovine serum (FBS) were obtained from Gibco (St Louis, MO, USA). A cell counting kit (CCK-8) was purchased from Biosharp (Hefei, China). Annexin V-FITC/PI staining kit was purchased from BestBio (Shanghai, China). Anti-MnSOD antibody, anti-Sirt3 antibody, anti-LC3A/B antibody and anti-Beclin1 antibody were purchased from Abcam (Beverly, MA, USA). Alexa Fluor 488-conjugated secondary antibody and 4′,6′-diamidino-2-phenylindole (DPI) were purchased from BEIJING BIOSS (beijing, China). MitoSOX Red mitochondrial superoxide indicator and 5,5′,6,6′-Tetrachloro-1,1′,3,3′-tetraethyl-imidacarbocyanine iodide (JC-1) for live-cell imaging was obtained from life technologies (San Diego, CA, USA).

Lipid peroxidation was evaluated by means of the TBARS (thiobarbituric acid-reactive substances) assay. Malondialdehyde (MDA) comes form Lipid peroxide degradation products which can be combined with thiobarbituric acid (TBA) to form pink products. MDA content was determined by using the MDA assay kit (Nanjing Jiancheng Bioengineering Institute, China) according to the manufacturer’s instructions. briefly, Serum of rats with various reagents blending were incubated in 95 °C water bath for 40 min and subsequently centrifuged at 3000 rpm for 10 min to obtain supernate which was estimated absorbance by spectrophotometry at 532 nm and the values expressed as nmol (mg protein)⁻¹. SOD activity, Antioxidant capacity, glutathione peroxidase and glutathione reductase ratio were determined by SOD activity assay kit, antioxidant capacity assay kit, glutathione peroxidase assay kit, glutathione reductase assay kit (Nanjing Jiancheng Bioengineering Institute, Nangjing, China). Simply, serum of rats blend with reagents for several minutes. Respectively, obtaining supernate was detected absorbance by spectrophotometry at 550 nm, 520 nm, 412 nm, 340 nm and the values expressed as percentage.

The LDH release, GSH content, caspase-3 activity and caspase-9 activity were determined using the LDH cytotoxicity kit, glutathione reductase assay kit, caspase-3 activity assay kit and caspase-9 activity assay kit (Nanjing JianCheng Bio Institute, Nanjing, China), respectively, according to the manufacturer protocol [18 (link),27 (link),28 (link)].