Nanophotometer p class p330 30

Colon, MLN, and spleen samples were isolated, snap frozen in liquid nitrogen, and stored at −80°C till analysis. Total RNA was extracted using ONE STEP RNA Reagent (BIO BASIC Inc., Markham, Ontario, Canada) according to manufacturer's protocol. RNA purity and concentration was assessed by NanoPhotometer® P-Class P330-30 (Implen, Munich, Germany). In order to purify RNA from all polysaccharides, including DSS, an additional purification step using 8 M LiCl was performed [29 (link)], followed by the standard genomic DNA purification step using Deoxyribonuclease I kit (Sigma Aldrich, St Louis, MO, USA). One microgram of RNA was used for cDNA synthesis by High Capacity cDNA kit with RNase Inhibitor (Applied Biosystems, Foster City, CA, USA). Real-time PCR was performed on CFX96 system (Bio Rad, Singapore) to assess relative expression of catalase (CAT), glutathione peroxidase 1 (GPx1), superoxide dismutase 1 (SOD1), HIF-1α, IL-1β, IL-2, and IL-6. Gene expression was normalized to HPRT1 gene. Primers list is given in the supplementary data (Table 1). Except for the primers for IL-6 gene published by Jeong et al. [30 (link)], all other primers were custom made using Primer 3 software. Messenger RNA expression was determined using SsoFast EvaGreen Supermix (Bio Rad, Singapore). Data are presented as mean ± s.e.m.

Thiobarbituric acid-reactive substance (TBARS) assay and plasma antioxidant capacity (FRAP) assay were performed according to the established protocol in our laboratory [27 (link), 28 (link)]. Arterial blood samples were collected from the femoral artery and centrifuged on 3500 rpm for 10 minutes, and serum samples were stored at −80°C until use. TBARS assay is based on the reaction of malondialdehyde (MDA), an end-product of lipid peroxidation, with TBARS. To correct for background absorption, the absorbance values at 572 nm were subtracted from those at 532 nm, which represent the absorption maximum of the TBA:MDA adduct [29 (link)]. Absorbance was monitored by NanoPhotometer® P-Class P330-30 (Implen, Germany). Results were compared with a standard curve with MDA and expressed as μM MDA equivalents. The antioxidant capacity of plasma was measured by the ferric reducing antioxidant power (FRAP) assay. In this assay, antioxidants are evaluated as reductants of Fe³⁺ to Fe²⁺, which is chelated by TPTZ (2,4,6-tris(2-pyridyl)-s-triazine) to form a Fe²⁺–TPTZ complex absorbing at 593 nm [22 (link)]. Results were compared with a standard curve with Trolox (TE), a water-soluble analogue of vitamin E, and expressed as μM TE equivalents.