Antibiotics antimycotics

Human MSCs from umbilical cord tissue were purchased from ATCC (Manassas, VA, USA). A total of three cell batches from different donors were used. MSCs were maintained in Minimum Essential Medium-alpha (MEM-α) (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (Atlas Biologicals, Fort Collins, CO, USA) and antibiotics-antimycotics (Genedirex, Taoyuan, Taiwan). MSCs cultured for five or six passages were used in all experiments. FD was purchased from Sigma-Aldrich (St. Louis, MO, USA). First, MSCs were cultured with various concentrations of FD (1, 3, or 5 µg/ml) for 24, 48, or 72 h. To evaluate the effect of FD on oxidative stress–induced cell death, FD (1 or 3 µg/ml) was treated with MSCs in the presence of 300 µM of hydrogen peroxide (Daejung Chemicals, Siheung-si, Korea) for 12, 24, or 48 h. Independently, MSCs prestimulated with 3 µg/ml of FD for 72 h was treated either with 500 µM for 6 h (for assessing ROS generation and apoptosis) or with 300 µM for 24 h (for measuring mitochondrial membrane potential). Cell viability was analyzed using a Cell Counting Kit-8 (CCK-8) (Dojindo Laboratories, Kumamoto, Japan) according to the manufacturer’s instructions. The detailed procedures for the characterization of MSCs are available in the Supplemental Materials and Methods.