Cultrex 96 well 3d spheroid cell invasion assay

A scratch wound invasion assay using IncuCyte Zoom and the Transwell invasion assay were used with Matrigel^® (BD Biosciences, Franklin Lakes, NJ, USA) diluted 1:4 with serum-free medium as the intervening invasive barrier. In addition, a 3D invasion assay was performed using the Cultrex 96-well 3D Spheroid Cell Invasion assay (Trevigen; Bio-Techne, Minneapolis, MN, USA), according to the manufacturer's protocol. In brief, 3×105 cells were suspended in 50 µl prechilled spheroid formation ECM, added to a Corning 96-well Clear Round Bottom Ultra Low Attachment Microplate (Corning Incorporated), centrifuged at 200 × g for 3 min at room temperature, and then incubated for 3 days to assemble into compact spheroids. Following the addition of 50 µl prechilled invasion matrix, the plates were centrifuged at 300 × g for 5 min at 4°C and incubated for 1 h at 37°C to promote gel formation. Culture medium containing the indicated concentrations of RA was then added into each well and the plates were incubated at 37°C in a 5% CO₂ incubator for between 3 and 5 days. Cells that invaded the surrounding matrix were observed using a phase-contrast inverted microscope (magnification, ×100) and images were captured every 24 h.