Tissue tearor 985370

The Myeloperoxidase (MPO) activity was used to determine stomach toxicity via a colorimetric assay, as this is a reliable marker for non-steroidal anti-inflammatory-drug-induced stomach lesions [36 (link),67 (link)]. Samples of the stomach were collected in 50 mM K₂HPO₄ buffer (pH 6.0) containing 0.5% hexadecyl trimethylammonium bromide (HTAB) and kept at −86 °C until use. Frozen samples were homogenized using a tissue turrax (Tissue-Tearor 985370, BioSpec Products, Bartlesville, OK, USA), centrifuged (2 min, 16,000× g, 4 °C), and the resulting supernatant was assayed using a spectrophotometer (Multiskan GO Microplate Spectrophotometer, Thermo Scientific, Vantaa, Finland) to determine MPO activity at 450 nm. The MPO activity of samples was compared to a standard curve of neutrophils. Briefly, 15 µL of sample was mixed with 200 µL of 50 mM phosphate buffer (pH 6.0) containing 0.167 mg/mL O-dianisidinedihydrochloride and 0.0005% hydrogen peroxide. The results were presented as MPO activity (number of neutrophils × 106/mg of tissue).

The superoxide anion production was determined by the reduction of the redox dye Nitroblue tetrazolium (NBT). Frozen knee joint tissue from mice was homogenized with 500 µL of saline using an ultra-turrax (Tissue-Tearor 985370, BioSpec Products, Bartlesville, OK, USA) and centrifuged (10 min, 3300× g, 4 °C); then, 50 µL of the homogenate was placed in 96-well plate, followed by the addition of 100 µL of nitro blue tetrazolium solution (1 mg/mL) (NBT, Sigma-Aldrich, St. Louis, MO, USA), and it was maintained at 37 °C in a warm bath for 5 min. The supernatant was removed, and the formazan that had precipitated was then solubilized by adding 120 µL of 2 M KOH and 120 µL of dimeltisulfoxide (DMSO). The optical density was measured using a microplate spectrophotometer reader (Multiskan GO Microplate Spectrophotometer, Thermoscientific, Vantaa, Finland) at 600 nm. The NBT reduction levels were corrected according to the total protein concentration and the results were presented as NBT reduction (OD/mg of protein) [68 (link)].