Apoptosis induction by germacrone

Eca109 and EC9706 cells were seeded in 6-well plates at a density of 1 × 10⁵ cells/well. After 24 h cultivation, cells were treated with different concentrations (0, 10, 20, 30 μg/mL) of germacrone for 24 h. Cells were then preprocessed and submitted to Annexin V/propidium iodide (PI) staining for FACS analysis according to the protocol described previously [23 (link)]. Briefly, cells were harvested by centrifugation (2000 rpm for 5–10 min) and resuspended in 300 μL of 1 × Binding Buffer. Subsequently, 5 μL of Annexin V-FITC and 5 μL of PI solution were added followed by incubation at room temperature for 15 min in the dark. Induction of apoptosis was measured by a Guava easyCyte flow cytometry (Millipore, MA, USA) and analyzed using FlowJo software.