8 isoprostane eia assay kit

Free isoprostane levels in the heart tissues were measured using 8-isoprostane EIA assay kit (Cayman Chemical). Briefly, tissues (20–30 mg) were homogenized in 0.1 M Tris buffer (pH 7.4) with 1 mM EDTA and 0.005% butylated hydroxytoluene. The homogenates were centrifuged at 8,000 g for 10 min at 4 °C. The supernatant was further diluted with EIA buffer and loaded into the strips pre-coated with mouse anti-rabbit IgG. The tracer (acetylcholinesterase linked to 8-isoprostane) and antiserum to 8-isoprostane were added to the samples in the wells and incubated for 18 h at 4 °C. After wash, the color was developed by incubating the plate in Ellman’s reagent for 2 h and the absorbance was measured with a spectrophotometer at 410 nm. Concentrations of isoprostane were calculated based on the standard curve.

Alkbh8^-/- and wt MEF cells were cultured in 6-well format and treated with 1.2 mM H₂O₂ in serum-free media for 1 h. After treatment media was replaced with complete growth media and incubated for 48 h in a humidified CO₂-incubator. Cells were harvested and lysed in RIPA buffer (50 mM Tris-HCl, pH 7.4, with 150 mM sodium chloride, 1% TritonX-100, 0.5% sodium deoxycholate, and 0.1% sodium dodecyl sulfate) supplemented with an EDTA-free protein inhibitor cocktail (Roche Diagnostics). Extracted protein was normalized using Bradford (BioRad) analysis according to manufacturer’s instruction. LPO was analyzed using the 8-Isoprostane EIA assay kit from Cayman Chemical Company (Ann Arbor, MI, USA). The 96-well assay plate was set up and run as suggested by the manufacturer, using an eight point standard curve run in duplicate. Protein samples were diluted in EIA buffer (supplied by the manufacturer) to a final concentration of 0.250 μg per well. Data reported comes from biological triplicates, with each sample assayed in duplicate. The 8-isoprostane sample concentrations from the mean absorbance were interpolated from a sigmoid standard curve (4 parameter logistic nonlinear regression) using Graph Pad software. A Student t-test was used to identify significant differences between groups at p < 0.05.