Lipid peroxidation mda assay kit

The relative Iron, ROS, MDA and GSH contents in cell lysates were detected using an Iron Assay Kit (#ab83366, abcam, USA), Total Reactive Oxygen Species (ROS) Assay Kit (#88-5930-74; Thermo Fisher Scientific, USA), Lipid Peroxidation (MDA) Assay Kit (#ab118970; Abcam, USA), and Glutathione (GSH) Assay Kit (#CS0260, Sigma, USA) according to the manufacturer's instructions, respectively.

The assay regarding the detection of reactive oxygen species (ROS), malondialdehyde (MDA) and superoxide dismutase (SOD) levels was performed to evaluate oxidative stress. In brief, 2 × 10⁶ HK-2 cells for each experiment were harvested, and subsequent procedures were carried out following the instruction of the cellular ROS assay kit (Abcam, Cambridge, MA, USA), lipid peroxidation MDA assay kit (Abcam), and SOD activity assay kit (Abcam). Finally, the outputs of these samples were measured by a microporous plate spectrophotometer (Bio-Rad).

Quantification of the oxidative stress marker malondialdehyde (MDA) was performed using Lipid Peroxidation (MDA) Assay Kit (Abcam, Cambridge, UK). In brief, TBA solution was incubated with standards and samples at 95℃ for 60 min. After being in ice bath for 10 min, the MDA-TBA adduct was analyzed at 532 nm wavelength using microplate reader (Bio-Rad).

We measured malondialdehyde (MDA) using the Lipid Peroxidation (MDA) Assay kit (#ab118970; Abcam) to fluorometrically measure lipid peroxidation according to manufacturer’s instructions. The colorimetric reaction was measured at 532 nm on a BioTek Synergy HT microplate reader (Biotek Instruments) and Gen5 Data Analysis Software (Biotek).

Immunoblotting was carried out as described [9 (link)]. Antibodies for beta-actin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), fatty acid synthase (FAS), alpha-smooth muscle actin (alpha-SMA), collagen 1A1 (Col1A1) and stearoyl-CoA desaturase 1 (SCD1) were from New England Biolabs GmbH (Frankfurt am Main, Germany). Manganese superoxide dismutase (MnSOD) antibody was from Thermo Fisher Scientific (Schwerte, Germany). Low density lipoprotein (LDL)-receptor antibody, diacylglycerol-O-acyltransferase (DGAT) 1 antibody and acetyl-CoA-carboxylase (ACC) antibody were from Novus Biologicals (Centennial, CO, USA). The 4-hydroxynonenal (4-HNE) and p53 antibodies were from R&D Systems (Wiesbaden-Nordenstadt, Germany). Immunoblots were quantified with Image J [29 (link)]. ELISA to measure alpha-fetoprotein (AFP) was from R&D Systems and serum was diluted 20-fold. Malondialdehyde levels were determined by a colorimetric assay from Abcam (Lipid Peroxidation (MDA) Assay Kit).

Cells (100, 000/well in 24-well plate) were treated with drug for 24 hours. ATP levels were measured by ATPlite Luminiescent Assay kit (Perkin Elmer, US). DNA was extracted using the DNEasy Mini Kit (Qiagen, US). Oxidative DNA and protein damage was measured by quantifying 8-hydroxy-2′-deoxyguanosine (8-OHdG) levels using the OxiSelect Oxidative DNA Damage ELISA Kit (Cell Biolabs) and by quantifying protein carbonylation levels using the Protein Carbonyl ELISA Kit (Enzo LifeSciences), respectively. Lipid peroxidation was measured using the Lipid Peroxidation MDA Assay Kit (Abcam) following the manufacturer’s protocol. The absorbance was read on a Spectramax M5 Microplate reader.

LVs were homogenized and centrifuged to determine the concentrations of the antioxidative and oxidative indicators. The level of superoxide dismutase (SOD) was determined via a colorimetric method using a SOD assay kit with WST-1 (BioVision, Inc., Milpitas, California, USA). The concentration of glutathione (GSH) was detected via the DTNB colorimetric method using the detection kit (Geno Technology, Inc., MO, USA). The level of malondialdehyde (MDA) was detected via the thiobarbituric acid colorimetric method using a Lipid Peroxidation (MDA) Assay Kit (Abcam, Cambridge, MA, USA). The activity of free 15-F2t-isoprostane was measured using a 15-Isoprostane F2t ELISA Kit (Neogen Co., Lexington, KY, USA). All procedures were conducted in strict accordance with the manufacturer's instructions. All samples were measured in triplicate, and the results were averaged.