Catalase assay kit

Catalase (CAT) activity was measured by ammonium molybdate method using Catalase assay kit (Nanjing Jiancheng Bioengineering Institute). Nitric oxide (NO) induction was measured by nitrate reductase method using Nitric Oxide assay kit (Nanjing Jiancheng Bioengineering Institute).

Honokiol was purchased from Meilunbio (Dalian, Liaoning, China), and its purity was ≥98%. The p16INK4α antibody, TBHP, and type II collagenases were obtained from Sigma-Aldrich (St. Louis, MO, USA). The primary antibodies of p-AMPK, PGC-1α, Mfn2, Drp1, Bnip3, and Bnip3L were acquired from Abcam (Cambridge, UK). The AMPK, SIRT3, and LC3 were supplied by CST (MA, USA). The Fis1 antibody was obtained from Genetex (Irvine, USA). The β-actin antibody, 4′, 6-diamidino-2-phenylindole (DAPI), malondialdehyde (MDA) assay kit, and total superoxide dismutase assay kit with NBT were purchased from Beyotime (Shanghai, China). Alexa-Fluor-488- and Alexa-Fluor-594-tagged second antibodies were obtained from Abcam. The catalase assay kit was obtained from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Compound C was obtained from Selleck (Houston, USA). The Bnip3L antibody, PGC-1α siRNA (sc-72151), siRNA transfection medium (sc-36868), and siRNA transfection Reagent (sc-29528) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). The reagents for cell culture were purchased from Gibco (Grand Island, NY, USA).

An oxidant-sensing fluorescent probe 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) was used to determine the total status of ROS as previously described (Li et al., 1999 (link); Cash et al., 2007 (link)). Other two reactive intermediates, H₂O₂ and nitric oxide (NO) were determined by phenol red (Pick and Mizel, 1981 (link)) and hemoglobin methods (Murphy and Noack, 1994 (link)), respectively. The SOD activity was measured based on the ability of the enzyme to inhibit the reduction of nitro blue tetrazolium (NBT) by superoxide radicals (Crouch et al., 1981 (link)). The CAT activity was measured using the catalase assay kit (Jiancheng, China).

The oxidation and antioxidative activities were determined according to the manufacturer’s recommended protocol. Nitric oxide (NO) induction was measured using the nitrate reductase method and the nitric oxide assay kit (Nanjing Jiancheng Bioengineering Institute). Catalase (CAT) activity was measured using the ammonium molybdate method and the catalase assay kit (Nanjing Jiancheng Bioengineering Institute). Peroxidase (POD) activity was measured using the absorbance method and the peroxidase assay kit (Nanjing Jiancheng Bioengineering Institute). Superoxide dismutase (SOD) activity was measured using the WST-1 method and the superoxide dismutase assay kit (Nanjing Jiancheng Bioengineering Institute).

The colon tissues of each group of mice were prepared according to the kit requirements to detect the levels of colonic oxidative stress markers. The concentrations of reduced glutathione (GSH) and oxidized glutathione (GSSG) (GSH and GSSG Assay Kit, S0053, Beyotime Biotechnology Co., Ltd., Shanghai, China), malondialdehyde (MDA) (Lipid Peroxidation MDA Assay Kit, S0131S, Beyotime), and catalase (CAT) (Catalase assay kit (Visible light), A007-1-1, Jiancheng) were detected using colorimetric kits. Glutathione peroxidase (GSH-Px) expression was detected using a Total Glutathione Peroxidase Assay Kit with NADPH (S0058, Beyotime). Superoxide dismutase (SOD) content was determined using a Total Superoxide Dismutase Assay Kit with WST-8 (S0101S, Beyotime). Protein concentration was determined with a BCA Protein Assay Kit (Beyotime).

Gastrodin was purchased from Chengdu Herbpurify CO., LTD (Chengdu, China). NaF was from Sinopharm Chemical Reagent CO., LTD (Shanghai, China). Sodium chloride injection (0.9%) was obtained from Shenyang North Pharmaceutical Factory (Shenyang, China). Ether and medical anhydrous EtOH (ethanol) were purchased from Beijing Chemical Works (Beijing, China). The antibody against Bax, caspase‐3 and caspase‐9 was from Proteintech Co. Ltd (Wuhan, China). The antibody to Bcl‐2 was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The Catalase Assay Kit was from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Other chemicals were provided by Beyotime Biotechnology Co. (Jiangsu, China). Mice MC3T3‐E1 cells (ATCC, Manassas, VA, USA) were grown in alpha‐MEM media (Invitrogen, Carlsbad, CA, USA) containing 10% FBS, 1% penicillin and streptomycin (Sigma, Taufkirchen, Germany).

Herein, 1 mg/mL Di-amino benzidine (DAB) and 1 mg/mL nitroblue tetrazolium (NBT) were used to stain the 5th to 8th rosette leaves from 7-week-old seedlings for H₂O₂ and O₂⁻, respectively [49 (link)], according to the previous method [16 (link)]. The measurements of H₂O₂, O₂⁻, and MDA were performed according to the previous studies [50 (link),51 (link)]. Briefly, leaf samples used for determination of H₂O₂ and O₂⁻ were extracted by 50 mmol/L phosphate buffer. Then, the supernatant was mixed with titanium sulphate and determined at 415 nm to calculate the concentration H₂O₂, while the supernatant was mixed with sulphanilic acid and α-naphthylamine and detected at the absorbance of 530 nm to determine the concentration of O₂⁻. MDA was determined by trichloroacetic acid and thiobarbituric acid, and the absorbance was determined at 532, 600, and 450 nm.
In addition, the activities of superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD) were detected using the Total Superoxide Dismutase Assay Kit (Nanjing Jiancheng, A001-1, Nanjing, China), the Catalase Assay Kit (Nanjing Jiancheng, A007-1, Nanjing, China), and the Peroxidase Assay Kit (Nanjing Jiancheng, A084-3, Nanjing, China), respectively.