3D Matrigel Invasion and Proliferation Assay

3D Laminin-rich Extracellular Matrix (lrECM) On-Top Cultures (referred to as 3D On-top Matrigel assays) were performed with an initial seeding density of 1x10³ PC3 cells per well of a 96-well plate and conducted as previously.^{45 (link)} For invasion assays, 15 000 PC3 or 25 000 DU145 cells were seeded overnight into Matrigel-coated (100 μg/ml in growth media) wells in a 96-well Image-lock plate (Essen BioScience Inc., Ann Arbor, MI, USA). Wounds were made through the monolayer of confluent cells using the 96-pin WoundMaker (Essen BioScience Inc.) according to the manufacturer’s instructions. Wells were washed twice with phosphate-buffered saline and matrigel (50 μl, 1 mg/ml in growth media) was added to each well and allowed to solidify before the initiation of imaging. Images were captured every 2 h for up to 48 h by the IncuCyte FLR live cell imaging system (Essen BioSciences Inc.). Wound closure kinetics were determined using the CellPlayer software module (Essen BioScience Inc.). For proliferation assays, cells were seeded as described for invasion assays and cell confluence determined using the same software. Proliferation was also assessed using the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher Scientific).