Annexin V apoptosis assay

Apoptosis was assessed using an Annexin V-FITC Apoptosis detection kit FITC (eBioscience), according to the manufacturer’s instructions. Briefly, cells were seeded in 24-well plates at a density of 4,600 cells per well. After 24 hours, the cells were treated with vehicle or leflunomide (at concentrations of 25, 50 and 100 µM). After 72 hours cells were trypsinised, washed in PBS and treated with fluorochrome-conjugated Annexin V and propidium iodide as indicated in the protocol. Cells were analyzed on the BD Accuri™ C6 flow cytometer and the data analyzed using the instrument software.