4.9. 3D Migration Assay

The cells were prepared as 3D spheroids in ULA plates, as previously mentioned. After treatment, the cells were pre-treated with mitomycin C (10 µg/mL) for 2 h and then the spheroids were transferred to the flat bottom microplates, thus allowing the cells to attach to the bottom. The cells were incubated for another 24 h. Images of the transferred spheroids were obtained using a LionheartFX live cell imager (Biotek, Winooski, VT, USA). The migration area was measured from the images using ImageJ software [56] and was calculated relatively to the spheroid area.