A8727

Cell viability was evaluated using an MTT assay. Then, 200 μL of IPEC-J2 cells (5 × 10 4 /mL) was added to each well of 96-well plates and cultured in DMEM/F12 with 10% FBS (complete medium) for 24 h. Then, the cells were cultured in DMEM/F12 without FBS (basal medium) for 12 h. After that, the cells were treated with H 2 O 2 at the concentrations of 0, 100, 200, 400, 600, 800, 1,000, 1,200, 1,400, and 1,600 μM or CRH (C3042, Sigma-Aldrich, St. Louis, Missouri, USA) at the concentrations of 0, 0.0001, 0.001, 0.01, 0.1, and 1 nM for 24 h, or the cells were treated with 0.01 nM CRH for 6, 12, 24, 36, or 48 h or 0.01 nM CRH and antalarmin (A8727, Sigma-Aldrich, St. Louis, Missouri, USA) at the concentrations of 0.001, 0.01, 0.1, 1, 10, and 100 μM for 36 h. Finally, the medium was discarded, and 10 μL MTT reagent and 90 μL basal medium were added. The cells were further cultured for 4 h, and the optical density (OD) was measured at a wavelength of 570 nm using an enzyme-linked immune detector (550, BIO-RAD, USA). The number of cells is presented as the percent of control cells.