Figure 3. Modified Transwell assay allowing real-time confocal imaging with defined barrier. A, Schematic of barrier used to image cells actively invading through pores of various sizes on the membrane of a Transwell insert. 1, Water-immersion objective (60×); 2, vitronectin (3 μg/ml); 3, Transwell invasion/migrating assay insert with pores of various size (3.0 μm, 5.0 μm, or 8.0 μm); 4, human glioma cells (D54-EGFP or U251-EGFP); 5, chemoattractant (10 ng/ml EGF in 17% serum-containing media); 6, migration assay buffer; 7, 3% agar; 8, vacuum grease. Temperature and pH were maintained at physiologic conditions and cells were imaged using a laser scanning confocal microscope. B, Confocal image of invading D54-EGFP cells (green) traversing 8.0 μm × 15.0 μm pores present on the membrane of a Transwell invasion/migration insert. Image represents a snapshot of cells midway through the process of invasion with the cell body present on either side of the membrane. Scale bar, 50 μm. C, Volume reconstructions of an individual glioma cell that successfully migrated from one side of the membrane, through the pore, to the other side of the membrane. Green, red, and blue lines represent the x-, y-, and z-axes, respectively. Image reconstructions were made from Z-stacks of 80 sections taken 800 nm apart and acquired every 20 min.