Mda assay kit

Jag2- or Sirt1-inhibited PASMCs were incubated in six-well plates and treated under normoxic or hypoxic conditions. The mitochondrial membrane potential was assessed with the JC-1 kit (Solarbio Life Sciences, Beijing, China) according to standard procedure. Fluorescent images were acquired using a fluorescence microscope (Olympus, Tokyo, Japan), and mitochondrial membrane potential depolarization was quantified by calculating the percentage of red/green fluorescence using ImageJ software. The mitochondria-targetingsuperoxide anionindicatorMitoSOX (5 mM, Yeasen Biotechnology, Shanghai, China)was used to assay mitochondrial ROS(mtROS) formation.After staining for 15 min, cells were washed with PBS and acell suspensionproduced with trypsin.Finally,mtROS production was detected by flow cytometry and quantified using FlowJo software.To detect oxidative stress in lung tissue, DHE staining was performed according to the manufacturer’s instructions. SOD, MPO, and MDA levels were assessed with a SOD assay kit (Sigma-Aldrich, St. Louis, MO), MPO assay kit (Invitrogen), and MDA assay kit (Sigma-Aldrich) according to the manufacturers’ instructions.

H₂O₂ content was detected using Fluorimetric Hydrogen Peroxide Assay Kit (Sigma-Aldrich). MDA content was detected using Lipid Peroxidation (MDA) Assay Kit (Sigma-Aldrich). ADP and ATP levels were detected using a EnzyLight™ ATP assay (BioAssay Systems).The levels of AsA, GSSG and GSH were detected as described previously^{4 (link),64 (link)}.

Serum creatinine and blood urea nitrogen (BUN) levels were measured using a biochemical analyzer (Hitachi, Osaka, Japan). Serum TNF-α, interleukin-6 (IL-6), and IL-1β levels were assessed using ELISA kits (R&D Systems, Minneapolis, MN, USA). Renal malondialdehyde (MDA) levels were measured using an MDA assay kit (Sigma-Aldrich, St. Louis, MO, USA).

The lipid peroxidation assay was determined by the MDA colorimetric reaction (532 nm) with thiobarbituric acid (TBA), using the MDA Assay Kit (Sigma-Aldrich, St. Louis, MO, USA) and following the manufacturer′s conditions.

Lipid peroxidation was determined by the reaction of its end product malondialdehyde (MDA) with thiobarbituric acid (TBA) according to the manufacturer’s protocol provided with the lipid peroxidation (MDA) assay kit (Sigma-Aldrich, Poznań, Poland).

To examine lipid peroxidation, MDA concentration (nmol/mg) in liver, lung and spleen was measured using the MDA assay kit (Sigma-Aldrich, St. Louis, MO, USA) as described previously (38 (link)). 10 mg of each sample were homogenized in 300 µl lysis buffer master mix using the TissueRuptor (Qiagen, Venlo, The Netherlands).