Smartspec 3000 spectrophotometer

MDA levels that indicated lipid peroxidation were measured spectrophotometrically (Smartspec 3000 spectrophotometer, Bio-RAD, USA) using thiobarbituric acid reactive substance assays [14 (link)]. Hepatic tissue was homogenized (Kontes Glass Co., USA) and 15% trichloroacetic acid (Sigma-Aldrich), 0.375% thiobarbituric acid (Alfa Aesar, USA), and 0.25 N HCl were added to 0.1 ml of the homogenate. The solution was then boiled for 15 min before cooling to room temperature. Then, after 10 min of centrifugation at 12,000 rpm, absorbance values were measured spectrophotometrically at 535 nm. Protein concentrations were determined using the Bradford assay and MDA activity expressed as ‘nmol/mg’ protein.
Superoxide dismutase (SOD) activity assays were performed using the pyrogallol method [15 (link)]. Tris-HCl (50 mM) and 1 mM pentetic acid buffer were used as reaction media, and pyrogallol (20 mM, 10 ml) in 10 mM HCl buffer and 10 ml 0.1 methylenediaminetetraacetic acid buffer were added. Homogenized hepatic tissue was then added to the reaction mixture and decreased pyrogallol absorbance values were monitored spectrophotometrically (Smartspec 3000 spectrophotometer, Bio-RAD, USA) at 420 nm. SOD activity was determined as the amount of enzyme that reduced the color change by 50% (i.e., 50% inhibition of pyrogallol auto-oxidation) and expressed as ‘U/mg’ protein [16 (link)].

Hepa1c1c7 cells (1 × 10⁵/mL) were seeded into 24-well culture plates and treated with a sample or the vehicle [0.5% dimethyl sulfoxide (DMSO), v/v] for 6 hours. Alternatively, the mucosa from the stomach, upper and lower small intestines, and large intestine were scraped off with a razor, and liver and kidney were taken from the euthanized mice, followed by storage at −80°C until use. Approximately 20 mg of each tissue were used for the total RNA extraction. Total RNA from cell cultures and animal tissues was isolated from cells using TRIzol reagent (Invitrogen), according to the manufacturer’s specifications. The amount and purity of RNA were assessed by spectrophotometry using a SmartSpec 3000 Spectrophotometer (Bio-Rad Laboratories (Hercules, CA)). Complementary DNA was synthesized using 1 μg of total RNA with an RNA polymerase chain reaction (PCR) Kit (avian myeloblastosis virus (AMV)). Thermal cycling was performed with a 7300 real time PCR system using SYBR green PCR mix (Applied Biosystems, Foster City, CA, USA), according to the manufacturer’s protocol. PCR conditions were as follows: 95°C for 3 minutes, 95°C for 10 seconds, and 60°C for 1 minute (mouse). The primers and sequences used are summarized in Table 1.