Spheroid Formation Assay Protocol

Forty-eight hours after transfection, 1 × 10⁴ of 0.05% trypsin-EDTA (HyClone, Cytiva)-detached cells in a final volume of 100 µL were seeded in Costar ultra-low attachment 96-well round-bottom plates (Cat No. 7007, Corning, Inc.) in complete medium (12 replicates). Spheroid formation was monitored and visualized using AxioObserver D1 microscope (10× lens; Carl Zeiss AG) and AxioVision LE software (Carl Zeiss AG) as previously described [32 (link)]. The area of spheroids was calculated using ImageJ (NIH) software. The results are presented as the percentage of spheroid area compared to controls (siNEG-treated cells).