DQ-collagen IV-based proteolytic activity assay

Mouse and human shSmyd5 cancer cells and control shLuc cells were serial diluted in the respective maintenance media, seeded in 350 μL/well of cold Matrigel and plated in a 24-well plate. Matrigel polymerization occurred after 30 min incubation at 37°C; 1 mL/well of maintenance medium (DMEM media containing 15% FBS, glutamine, 2-mercaptoethanol, and non-essential amino acids) was added and replenished every other day. The proteolytic activity of the Matrigel embedded cells was ascertained by first washing the cells with warm PBS and then incubating overnight in the presence or absence of DQ-collagen IV (10 μg/mL) in serum free, phenol red free DMEM at 37°C. DQ-collagen IV is a heavily fluorescein labeled collagen IV substrate which yields green fluorescence upon cleavage by matrix metalloproteinases (MMPs). The fluorescence resulting from the DQ-collagen IV cleavage was visualized using a Leica DMIRB fluorescence microscope equipped with the SPOT RT3 camera and the SPOT advanced software. The cell nuclei were counterstained with Hoechst or DAPI dye.