Trans-Well Migration and Invasion Assay

The 8 μm pore trans-wells were purchased from BD Biosciences (Franklin Lakes, NJ, USA). CL1-5-F4 cells were treated with different concentrations of imipramine for 48 hr and then cells were collected into the upper channel of trans-wells at the number of 5×10⁵, allow 24 hr migration period of CL1-5-F4 cells. Alternatively, to test invasion ability, the upper channel of the trans-well was pre-coated with matrigel. Transwell membranes were then fixed (methanol and acetic acid 3:1) and stained by 0.5% crystal violet. The light microscope (Nikon ECLIPSE Ti-U) was used to photograph the migration and invasion cells at ×100 on the trans-well membrane. The number of migration and invasion cells was calculated using Image J software version 1.50 (National Institutes of Health, Bethesda, MD, USA) (19).