Malondialdehyde tetra butyl ammonium salt mda

All reagents and chemicals were of analytical grade and applied without further purification. HPLC-grade deionized water was used during all experiments.
α-KG, 5-HMF, NASeLM, NALM, and glucose were supplied by CYL-Pharma (Lassnitzhöhe, Austria). NaCl, KCl, KH₂PO₄, MgSO₄, CaCl₂, NaHCO₃, and dextrose were obtained from Roth (Karlsruhe, Germany). Acetonitrile, ammonium acetate, 1-butanol, ethanol, HPLC-grade water, hydrogen peroxide, and HCl were obtained from Merck (Darmstadt, Germany). Albumin from human serum, angiotensin 1-7 acetate salt hydrate, butylated hydroxytoluene (BHT), 2,4-dinitrophenylhydrazine (DNPH), ethyl acetate, guanidine-hydrochloride, malondialdehyde tetra-butyl-ammonium salt (MDA), 2-thiobarbituric acid (TBA), trichloroacetic acid (TCA), and tris(hydroxymethyl)aminomethane (Tris) were obtained from Sigma-Aldrich (Vienna, Austria).

The human brain vascular pericyte (HBVP) cell line were cultured at 37°C in 5% CO2, 95% air in Pericyte medium (PM) (ScienceCell Inc.) containing 4% FBS, 1% Pericyte growth supplement (PGS), 100 U/mL penicillin and 100 μg/mL streptomycin (ScienceCell Inc.). HBVP are characterized by using antibody specific to | |-smooth muscle actin and are negative for HIV-1, HBV, HCV, mycoplasma, bacteria, yeast, and fungi. In all experiments, cells were grown to 70-90% confluence and subjected to a maximum of 20 cell passages. For the metabolic activity assay, 1×10⁴ HBVP were incubated with 0.5 μg of human soluble Aβ 1-40 monomers (cat. num.: AS-24236-5, AnaSpec) and/or 1 mM of malondialdehyde tetrabutylammonium salt (MDA; cat. num.: 63287, Sigma Aldrich) for 24 h prior to the absorbance based “XTT Cell viability” assay kit (cat. num.: 9095, Cell Signaling). The cell viability assays were performed accordingly to the manufacturer's protocol.

The lipid peroxidation protocol previously described was modified for this study (Jambunathan, 2010 (link)). Barley seeds (approx. 300 mg) from each stage of malting was ground to fine powder with pestle and mortar using liquid nitrogen. Finely ground powder was transferred to a tube containing 1.0 mL 0.1% TCA, vortexed, and allowed to sit at room temperature for 10 min. The extracts were centrifuged at 16,000g for 10 min. The supernatant was collected, and 1 mL supernatant was mixed with 500 μl of 20% TCA and 500 μl of 0.5% TBA and vortexed. Malondialdehyde tetrabutylammonium salt (MDA) (Sigma) was used for preparing the standards. The 2 mL tubes were sealed with no-pop tops before placing in water bath at 85°C for 1 h. Tubes were then cooled on ice. Following centrifugation at 16,000g for 5 min, 200 μl of sample/well were transferred to 96-well plate. The plate was placed on a shaker at 1,000 rpm for 1 min. The absorbance at 532 and 600 nm against a blank solution (1 ml of 0.1%TCA + 500 μl of 20%TCA and 500 μl of 0.5% TBA) was read using a plate reader (Synergy H1, Biotek, Winooski, VT, United States). A₆₀₀, the non-specific absorbance was subtracted from the values for A₅₃₂. The concentration of MDA in the seed samples were estimated using the standard curve.