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High Content Analysis of an Automatable 3-Dimensional Cell Invasion Assay
Renee L. Herber1, Jennifer A. Fronczak1, Timothy Baranowski2 and Keren I. Hulkower1
1Platypus Technologies, LLC and 2Molecular Devices Inc.
The ability of cells to invade through an extracellular matrix is a hallmark event in the metastasis of tumor cells. Understanding the signaling pathways involved in invasion is crucial for discovering new targets to develop anti-metastatic drugs for treating cancer. Because cell invasion is a phenotypic or whole-cell event, it can be challenging to conduct and quantitatively analyze such cell-based assays in a screening format. A novel invasion assay platform that is amenable to automated liquid handling now allows for both an increased capacity and decreased hands-on time for screening assays. This assay utilizes a centrally located self-dissolving biocompatible gel (BCG) to form a uniformly sized, cell-free detection zone on a collagen I coated cell culture surface. Cells are seeded into 96-well plates and pattern in an annular monolayer surrounding the BCG. Once the BCG dissolves, an overlay of collagen I is applied to the assay wells and cells can invade in 3-dimensions into the detection zone previously occupied by the BCG. Inhibitors, such as Cytochalasin D, may be added to the assay wells in the media covering the collagen I overlay. This assay format allows visual assessment of cell invasion throughout the duration of the experiment. Cells may be fixed and treated with multiple stains, including DAPI to visualize nuclei and TRITC-phalloidin to observe F-actin. Such stains enable flexible data capture by either enumerating invading cells or by calculating the area of closure within the detection zone. Here we demonstrate successful analysis of the Platypus Technologies Oris™ Pro Collagen I Cell Invasion Assay using the Molecular Devices ImageXpress® Micro Widefield HCS System and MetaXpress® and AcuityXpress™ Software and demonstrate robust and reproducible quantification of HT-1080 cell invasion in 3-dimensions.
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