Glutathione peroxidase gpx assay kit

A commercially available glutathione peroxidase (GPx) assay kit (Cayman Chemical Company) was used to measure plasma GPx activity according to the manufacturer’s instructions. Glutathione (GSH) and total GSH were measured using GSH colorimetric assay (BioVision Incorporated, CA, USA). Oxidised glutathione/glutathione disulfide (GSSG) was calculated by subtraction of GSH from total GSH.

Dulbecco's modified Eagle's medium (DMEM), trypsin, fetal bovine serum (FBS), penicillin, streptomycin, and fungizone were obtained from Hyclone (Logan, UT, USA). 1-Nitropyrene (1-NP), 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT), phosphate-buffered saline (PBS), dimethyl sulfoxide (DMSO), and other reagents of analytical grade were obtained from Sigma-Aldrich (St. Louis, MO, USA). Tetraethylbenzimidazolylcarbocyanine iodide (JC-1), catalase (CAT) assay kit, superoxide dismutase (SOD) assay kit, and glutathione peroxidase (GPx) assay kit were obtained from Cayman (Ann Arbor, MI, USA). Mitochondrial permeability transition pore (MPTP) assay kit was obtained from BioVision (Milpitas, CA, USA). Primary antibodies for the detection of AIF, cytochrome c, Bcl-2, Bcl-xL, Bad, Bax, Bid, HO-1, Nrf2, P-AMPK, AMPK, COX-IV, GAPDH, and β-actin were obtained from Santa Cruz (Santa Cruz, CA, USA). Secondary antibodies for horseradish peroxidase- (HRP-) conjugated mouse anti-rabbit IgG or goat anti-mouse IgG were purchased from Jackson ImmunoResearch Laboratories (Baltimore, MD, USA). Dissolve 1-NP in DMSO, in which the final concentration was not higher than 0.05% (v/v).