Elisas

To assess the effect of IRW on cell viability, a 3-(4,5-dimethylthiazol-2-yl)-2,5-biphenyl tetrazolium bromide (MTT) test was used to identify metabolically active viable cells. Briefly, HUVEC cells were cultured for 24 hours at 37°C and then treated with different concentrations of IRW for 2 hours. Subsequently, 1 μg/ml LPS was added to the cultures, which were incubated for 16 hours. Next, a 5 mg/ml MTT solution was added; the mixtures were incubated for 3 hours at 37°C and then stored overnight in sodium dodecyl sulphate (SDS) buffer (10%) containing 0.01 M HCl. A spectrophotometer (TECAN, Austria) was used to detect the absorbance in each well at 570 nm. These experiments were conducted in triplicate. Additionally, the levels of IL-8 and TNF-α in the culture medium were measured using ELISAs according to the user's manuals (Nanjing Jiancheng Bioengineering Institute, Nanjing, Jiangsu, China).

The activity of glutathione peroxidase (GSH-Px), superoxide dismutase (SOD), malondialdehyde (MDA) and catalase (CAT) in serum, liver, heart, and intestine samples and IPEC-J2 cells were detected using ELISAs according to the manufacturer’s instructions (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). The absorbances were measured at 412 nm for GSH-Px, 550 nm for SOD, 532 nm for MDA, and 405 nm for CAT. The minimal detection thresholds were 20 U/mL for GSH-Px, 5 U/mL for SOD, 0.5 nmol/mL for MDA and 0.2 U/mL for CAT. For each assay, the intra-assay coefficient of variation (CV) was < 5%, and the inter-assay CV was < 6%. Six samples per tissue were tested for each treatment group, and each sample was assayed in triplicate.