Time-Lapse Imaging of 3D Tumor-Stromal Coculture

NHDFs, F-NHDF, HPMFs, F-HPMFs, CC-NHDFs or CC-HPMFs were plated onto the collagen-coated insert and overlayed with a Matrigel/MDA-MB-231 cell suspension as described above. 2D images at 4X and 10X magnification were acquired through an Olympus CK2 microscope housed in an incubator at 37°C and 5% CO₂. For 3D analyses of coalescence with DIC optics, a lid with a glass insert was used to cover the dish. 3D cultures were imaged through a 20x objective using Differential Interference Contrast (DIC) optics on a Zeiss Axioplan 2 microscope with a motor-driven stage synchronized to a Zeiss AxioCam MRc5 IEEE 1394 color CCD camera and an LED light source. The microscope was housed in an incubator at 37°in 5% CO₂. A z-series of optical sections was acquired through the preparation at 10 μm increments and this process repeated every 10 minutes for up to 5 days. The z-series was imported into J3D-DIAS4.2 and saved in movie format for 3D reconstructions, as previously described [13 (link), 14 (link), 22 (link), 23 ].