Spectramax i3x platform plate reader

Liver samples were homogenized in Buffer A (50 mM NaPO4, 1 mM EDTA, pH 7.5) as in previous experiments. The supernatant (50 μL) was vortexed with 6.25 μL butylated hydroxytoluene (BHT) (4 mM in ethanol), and 50 μL ortho-phosporic acid (0.2 M). Thiobarbituric acid reagent (6.25 μL; 0.11M in 0.1M NaOH) was added and the samples were vortexed for 10 s. The samples were placed in a heating bath for 45 min at 90 °C followed by 2 min on ice, and 5 min at room temperature. N-Butanol (500 μL) and saturated NaCl (50 μL) were added to each sample before another 10 s of vortexing. Samples were centrifuged at 13,700× g for 2 min and 300 μL of the top phase were added to the wells. The plate was thereafter read at 532 nm (25 °C) using a SpectraMax i3x platform plate reader (Molecular Devices, China). Systemic TBARS were also measured by using 50 μL of plasma instead of 50 μL of liver homogenate supernatant.

Liver samples (~100 μg) were homogenized in 2 mL methanol, and 1 mL chloroform was added to the lysates. Lysates were vortexed briefly before it was centrifuged at 3000× g for 1 min to allow for separation. Of note, for samples that did not separate 100 μL saturated NaCl was added and such samples re-centrifuged. The bottom layer of the samples was pipetted into new microtubes and left open overnight at 4°C to dry out. On the second day 700 μL cyclohexane was added to each of the microtubes after it was vortexed. Subsequently, 200 μL of sample was added to a 96-well plate and the absorbance was read on a SpectraMax i3x platform plate reader (Molecular Devices, China) set at 25 °C and 232 nm using cyclohexane as a blank. All samples were assayed in triplicate.