Detection by ELISA of laminin released by endothelial cells from a collagen coated layer of Matrigel

Abstract

We have developed an in vitro model system quantitating the degradation of extracellular matrix by endothelial cells. Collagen is layered over a reconstituted basement membrane matrix (Matrigel) and the release of laminin contained in the Matrigel into the media by endothelial cells is quantitated by ELISA. The cell-specific release of laminin is consistent and reproducible. Incubating an equivalent number of dead endothelial cells in the same manner results in no release of laminin relative to media controls. The cell-specific laminin release is abolished by removing the exogenous growth factor component of the media and plating the cells in media plus 10% serum. Cells plated onto wells coated with collagen alone do not release laminin into the media, indicating that no de novo synthesis and release of laminin occurs during the time frame of the experiment, and that some factor or combination of factors present in the media supplement is essential for the endothelial cells to release laminin degrading enzymes.