Colorimetric activity assay kit

The accumulation of MPO in colon tissues was measured as a marker of neutrophil influx into the colon. Colon tissues were thawed and homogenized in lysis buffer. Subsequently, the homogenates were centrifuged at 1500 ×g for 15 min, and the resulting supernatants were assayed for MPO assay using the colorimetric activity assay kit (Sigma, St. Louis, MO, USA) according to the manufacturer's recommended protocol.

Colonic tissue homogenates were used to measure MPO and MDA activity. MPO activity was measured using a colorimetric activity assay kit (Sigma-Aldrich, St. Louis, MO, USA). In brief, the tissues were homogenized in the buffer solution provided with the kit, and the assay was performed according to the manufacturer’s instructions. Colorimetric change was measured on a microplate reader (BioRad, Hercules, CA, USA) using 412 nm absorbance, and the results were used to calculate the MPO concentration in each sample as per the manufacturer’s instructions. Similarly, the lipid peroxidation in colonic tissue was investigated using a commercial kit (Abcam, Cambridge, UK) that assesses MDA levels though a method dependent on thiobarbituric acid (TBA). The assay was performed according to the manufacturer’s protocol where TBA was added to the homogenate supernatant, and the samples were boiled for 60 min at 95 °C before the absorbance was assessed at 532 nm using a spectrophotometer (BioRad, Hercules, CA, USA).