Three-dimensional tumor spheroid-based functional assays: migration and invasion. (a) Tumor spheroid-based migration is illustrated in a cartoon: a single day 4 tumor spheroid was transferred to each well of extracellular matrix (ECM)-coated 96-well flat-bottomed plate using a multichannel pipette. (b) Representative brightfield images of U-87 MG and KNS42 cell migration (48 h) are shown. (c) Tumor spheroid invasion into Matrigel is illustrated in a cartoon: a single tumor spheroid per well was embedded in Matrigel. (d) Representative images of a typical U-87 MG 'starburst' invasion pattern obtained with a Celigo cytometer. The Cell Counting Confluence application (1/16 field of view/well) produces a segmentation analysis that identifies the area occupied by invading cells. (e) Representative images obtained using a conventional inverted microscope show the U-87 MG invasion at low magnification and details of invadopodia advancing into the matrix shown at higher magnification. Scale bar: 100 μm.