A Medium-Throughput System for In Vitro Oxidative Stress Assessment in IPEC-J2 Cells.
The feed industry continuously seeks new molecules with antioxidant capacity since oxidative stress plays a key role in intestinal health. To improve screening of new antioxidants, this study aims to set up an assay to assess oxidative stress in the porcine small intestinal epithelial cell line IPEC-J2 using plate-reader-based analysis of fluorescence. Two oxidants, H2O2 and menadione, were tested at 1, 2 and 3 mM and 100, 200 and 300 µM, respectively. Trolox (2 mM) was used as the reference antioxidant and the probe CM-H2DCFDA was used to indicate intracellular oxidative stress. Cell culture, reactive oxygen species (ROS) production and assessment conditions were optimized to detect a significant ROS accumulation that could be counteracted by pre-incubation with trolox. Menadione (200 µM) reproducibly increased ROS levels, H2O2 failed to do so. Trolox significantly decreased intracellular ROS levels in menadione (200 µM)-exposed cells in a consistent way. The system was further used to screen different concentrations of the commercially available antioxidant ELIFE®. Concentrations between 100 and 200 ppm protected best against intracellular ROS accumulation. In conclusion, the combination of CM-H2DCFDA fluorescence analysis by a plate-reader, trolox as a reference antioxidant and 200 µM of menadione as a stressor agent, provides a replicable and reliable medium-throughput setup for the evaluation of intracellular oxidative stress in IPEC-J2 cells.
['Ayuso|Miriam|M|', 'Van Cruchten|Steven|S|0000-0002-3144-1840', 'Van Ginneken|Chris|C|0000-0002-1622-9120']
D000821:Animal Feed D000818:Animals D000975:Antioxidants D002460:Cell Line D002839:Chromans D004847:Epithelial Cells D005452:Fluoresceins D005456:Fluorescent Dyes D057166:High-Throughput Screening Assays D006861:Hydrogen Peroxide D007421:Intestine, Small D016877:Oxidants D017382:Reactive Oxygen Species D013552:Swine D024483:Vitamin K 3
in vitro oxidative stress IPEC-J2 antioxidants fluorescence menadione ROS