Mda assay kit

Western blot antibodies were purchased from Cell Signaling Technology and Novus Biologicals; LY294002 was obtained from Sigma-Aldrich. Mouse cytokine ELISA Kits were purchased from R&D Systems. MaR1, MDA assay kit, MaR1 Elisa kit, and GPX activity assay kit were purchased from Cayman Chemical. Boc2 was obtained from Phoenix Pharmaceuticals, Inc. Lactate dehydrogenase (LDH) kit was purchased from Thermo Scientific. CCK-8 kit was obtained from Dojindo Molecular Technologies, Inc.

H₂S and MDA levels in bladder tissues were assessed with commercial kits[H₂S assay kit (kit code: E-BC-K355-M; Elabscience Biotechnology Co.Ltd, Wuhan, China) and MDA assay kit(kit code:700870; Cayman Chemical, Ann Arbor, MI, USA)] based on the instructions. Approximately half of the bladder was homogenized in RIPA. The homogenate was centrifuged at 10,000g and 1,600g for 10min at 4°C to collect the supernatant. The absorbance at 665nm for H₂S measurement and 540nm for MDA measurement was calculated via a microplate reader(Thermo Scientific, Waltham, MA, USA). Bladder H₂S and MDA concentrations are expressed as nmol per mg protein by determining protein concentrations in bladder tissue samples using the BCA assay kit.

Immediately before euthanasia, blood samples were collected from the previously cannulated left carotid artery. Serum was obtained by centrifugation at 1500× g for 10 min. Alanine aminotransferase (ALT), aspartate aminotransferase (AST), indirect bilirubin, direct bilirubin, total bilirubin, total protein, albumin, globulin, the albumin/globulin ratio, creatinine, urea, triglycerides (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), alkaline phosphatase, γ-glutamyl transpeptidase (GGT), sodium, calcium, magnesium, chloride, and potassium levels were measured using an automated biochemical analyzer (Roche Cobas Integra 400 plus). Low-density lipoprotein cholesterol (LDL-C) and very-low-density lipoprotein cholesterol (VLDL-C) were calculated according to the Friedwald formula [5 (link)]. Serum oxLDL, nitrotyrosine (NT), soluble VCAM-1 (sVCAM-1), soluble ICAM-1 (sICAM-1), IL-1β, and IL-6 levels were measured using an enzyme-linked immunosorbent assay (ELISA; BD Biosciences, San Jose, CA, USA). Malondialdehyde (MDA) levels were measured using an MDA assay kit (Cayman Chemical, Ann Arbor, MI, USA). Estradiol, thyroid-stimulating hormone (TSH), and free triiodothyronine (T3) were measured using a chemiluminescence immunoassay. Hepatic triglyceride content was measured using a previously described method [19 (link)].

Immediately after MVB withdrawal, blood samples were collected from the previously cannulated left carotid artery. Serum was obtained via centrifugation at 1500× g for 10 min. Alanine aminotransferase, aspartate aminotransferase (AST), creatinine, urea, triglycerides, total cholesterol, and HDL-C levels were measured using a semi-automated biochemical analyzer (Aker BIO-200). LDL-C and VLDL-C were calculated according to the Friedwald formula [5 (link)]. Serum NT, oxLDL, sVCAM-1, sICAM-1, IL-1β, and IL-6 levels were measured using enzyme-linked immunosorbent assays (BD Biosciences, San Jose, CA, USA). Malondialdehyde levels were measured using an MDA assay kit (Cayman Chemical, Ann Arbor, MI, USA).

In serum samples, total antioxidant capacity was determined using an Antioxidant Assay Kit, which assesses the combination of both small molecule and protein antioxidants (Cayman Chemical Company, Michigan, USA); catalase activity using a Catalase Assay Kit (Cayman Chemical Company, Michigan, USA); and malondialdehyde (MDA) using a MDA Assay Kit (Cayman Chemical Company, Michigan, USA).