Standardization and automation of the 3-D invasion assay.

<p>A) Schematic diagram (top panel) of the 3-D invasion assay with tooled plate: (a) The cell-collagen mixture is dotted into the 2 mm in diameter pits in the center of each well of the tooled 96-well plate, creating spheres that occupy a volume of 4.18 mm³; (b) protruding cell-collagen hemispheres are covered with collagen; (c) medium containing compounds is added and plate is incubated for 18 hours; and (d) invaded cells beyond pit boundary are counted. Black dots represent cells. Diagram not to scale. An image of a section of a tooled plate with a pit in the center of each well is shown (bottom panel). B) Inhibition of collagen contraction via dialdehyde dextran: HT1080 cells were mixed with collagen with or without the addition of dialdehyde dextran before overlaying with collagen. Phage contrast images were taken at time 0 and 18 hours (left panel, Scale bar = 250 μm) and 18 hours in the tooled plate (right panel, Scale bar = 100 μm). Red arrows indicate the edge of the pits. Red arrowheads indicated the edge of the cell-collagen spheres. C) Phase contrast and fluorescent images of invaded HT1080 cells in the tooled plate stained with Hoechst dye. Scale bar = 100 μm. D) Automated quantification: Both phase contrast and fluorescent images (a & b) of HT1080 cells following invasion were acquired using a Nikon TE-2000s controlled by the NIS Elements imaging software. A threshold of the phase contrast image of the first well was adjusted based on the contrast to create two binary layers, one inside the pit (highlighted in black) and one outside of pit (highlighted in pink) (c). The binary layer outside the pit (pink area) was copied to form a region of interest (ROI), which was then applied to the Hoechst image (d). The invaded cells within the ROI were then automatically counted with the counted cells appearing purple in color (e).