T aoc assay kit

Lipopolysaccharides (LPSs, from Escherichia coli serotype 026: B6) were from Sigma Chemical Co. (St. Louis, MO, USA). The CCK-8 Kit, ROS Assay Kit, T-AOC Assay Kit, Lipid Peroxidation MDA Assay Kit, Griess Reagent, ELISA kits for TNF-α, IL-1β, IL-6, and IL-10 were from Beyotime Biotechnology Co. (Shanghai, China). Anti-TLR4 antibody (ab13556), anti-NF-κB p65 antibody (ab32536), anti-NF-κB p65 (phospho S536) antibody (ab76302), anti-beta actin antibody (ab8227), and secondary antibody (ab97051) were from Abcam (Cambridge, MA, USA). Human/Mouse/Rat Keap1 antibody (MAB3024) and Human/Mouse Nrf2 antibody (AF3925) were from R&D Systems (Minneapolis, MI, USA). IκBα (44D4) Rabbit mAb and Phospho-IκBα (Ser32) (14D4) Rabbit mAb were from Cell Signaling Technology (Beverly, MA, USA).

The total antioxidant capacity of cells was measured using the ABTS (2,2-diazo-di(3-ethyl-benzothiazol-6-sulfonic acid) diammonium salt) method. After cell lysis, the supernatant was obtained and diluted. The diluted supernatant was then added to the pre-prepared working reagent following the instructions provided in the T-AOC assay kit (Beyotime, Shanghai, China). The absorbance at 734 nm was measured (n = 4), and the total antioxidant capacity of the sample was calculated based on a standard curve.

The state of redox in xenograft tumors and OS cells were determined by measuring the amount of ROS with ROS assay kit (S0033, Beyotime, China), the amount of malondialdehyde (MDA) with Lipid peroxidation MDA assay kit (S0121, Beyotime, China), the amount of total antioxidant capacity (T-AOC) with T-AOC assay kit (S0121, Beyotime, China), and the amount of superoxide dismutase (SOD) with Total SOD assay kit (S0109, Beyotime, China) according to manufacturers’ instructions. The level of ROS was detected by CytoFlex S (Beckman, USA) and the others were detected by Infinite pro 200 (Tecan, Switzerland). The levels of MDA and T-AOC were expressed as μmol/g protein. And the level of SOD was expressed as U/g protein.

Reactive oxygen species (ROS) were detected by examining the immunofluorescence of the CM‐H2DCFDA diacetate probe (# C6827, Invitrogen). The hPASMCs were transfected as shown and incubated for 24 h in six‐well plates (1 × 10⁶ cells/well) in SMCM and grown to 80% confluence. After serum deprivation for 12 h, hPASMCs were pretreated with or without PDGF‐BB (20 ng/mL), and when required ontuxizumab (10 µg/mL) was added for 24 h. The cells were washed with FBS‐free SMCM and incubated with CM‐H2DCFDA (10 µM) for 1 h at 37°C. Images were captured using a confocal microscope (TCS‐Leica SP5II, Germany). The images were quantitated using the ImageJ software. The hydrogen peroxide detection assay with CBA,⁵⁴ the superoxide anion detection with the superoxide assay kit, and the total‐antioxidant capacity (T‐AOC) detection with the T‐AOC assay kit (Beyotime, China) was performed according to the manufacturer's instruction.⁵⁵

Superoxide production in myocardial tissue was detected by fluorescence microscopy using the fluorescent probe dihydroethidium (DHE). Myocardial tissue sections (5 μm) were prepared and subsequently incubated (30 min, 37°C) in Krebs-HEPES buffer (mM components: NaCl 99, KCl 4.7, MgSO₄ 1.2, KH₂PO₄ 1.0, CaCl₂ 1.9, NaHCO₃ 25, glucose 11.1, NaHEPES 20; pH 7.4) and DHE (2 μM) in a dark room. The slides were examined using a Nikon TE2000 inverted microscope (Nikon, Tokyo, Japan) with excitation and emission wavelengths of 480 and 610 nm, respectively.
The left ventricular tissue was homogenized with precooled PBS in the ratio of 1 : 9 and then centrifuged at 4°C and 1600 rpm for 10 min to obtain the supernatant. BCA protein concentration determination kits were used to determine the protein concentration for subsequent quantitative calculation. Malondialdehyde (MDA) levels in the myocardium were measured using the thiobarbituric acid method (Beyotime) with a lipid peroxidation and assay kit. The total antioxidant capacity (T-AOC) of the myocardium was measured by the T-AOC assay kit and plasma iron-reducing capacity method (Beyotime). The activities of total SOD, Cu-Zn/SOD, and Mn-SOD in myocardium were measured using the WST-1 (2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-)-2H-tetrazole method (Beyotime).