Total superoxide dismutase assay kit with nbt

Cell pretreatment was the same as previously indicated in the CCK-8 assay. Total superoxide dismutase (SOD) activity was measured using a Total Superoxide Dismutase Assay Kit with NBT (S0109, Beyotime) following the manufacturer’s protocol. After 24-h treatment with DEX and EGCG, osteoblasts were washed twice with cold PBS, then lyzed in PBS by pulse sonication on ice, and subsequently centrifuged at 13,000 g at 4°C for 10 min. The supernatant was then transferred to a fresh tube. The sample protein concentration was measured and adjusted to 1 μg/μL. A 20 μL volume of the sample or SOD assay buffer (blanks) was added to a 96-well plate, along with 160 μL of NBT/enzyme working solution and 20 μL of reaction initiation working solution, to all wells except for Blank2. The plate was incubated at 37°C for 30 min. SOD activity was calculated after reading absorbance at 560 nm on a microplate reader.

For the determination of metabolic products and enzymatic activities, three pools of samples were prepared by pooling ~15 mg frozen mantle of five individuals at each sample time of each subspecies. Mixed tissue was added to an excess volume (9×) of precooled saline and ground using a homogenizer on ice. Precipitates were removed by centrifuging for 10 min at 2500 rpm and 4°C. Supernatants were diluted ninefold using saline and stored at −80°C.
The content of total protein (Total protein quantitative assay kit) and malondialdehyde (Malondialdehyde (MDA) assay kit (TBA method)), and the enzymatic activities of pyruvate kinase (Pyruvate kinase (PK) assay kit) and total ATPase (ATPase assay kit) was measured using the corresponding kits purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China), and total superoxide dismutase (SOD) was determined by Total Superoxide Dismutase Assay Kit with NBT (Beyotime, Shanghai, China). Assays performed following the corresponding manufacturer's protocols (Buege & Aust, 1978; Hopkirk & Bloxham, 1980; Sun, Oberley, & Li, 1988), and absorbance values were determined using a Varioskan Flash Multimode Reader (Thermo Fisher Scientific, Waltham, MA, USA).

Walnut meal provided by Hebei Yangyuan Zhihui Beverage Co., Ltd. (Hengshui, China). 1,1-diphenyl-2-picryl hydrazyl (DPPH), 2,2′-Azinobis-(3-ethylbenzthiazoline-6-sulphonate) (ABTS•+), Trypsin, and alkaline protease were purchased from Beijing Biotopped Technology Co. (Beijing, China). A BCA Protein Assay Kit (Biorigin, Louisville, KY, USA, BN16075) was purchased from Biorigin (Beijing) Inc. (Beijing, China). A Reactive Oxygen Species Assay Kit, a Catalase Assay Kit, and a Total Superoxide Dismutase Assay Kit with NBT were purchased from the Beyotime Institute of Biotechnology (Shanghai, China).

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).