Invasion Assays and Matrix Metalloproteinases: Quantification of Cellular Invasion Using Propidium Iodide Labeling and Confocal Laser Scanning Microsc

Matrix metalloproteinases are believed to play a major role in cellular invasion and tumor metastasis. Therefore, to understand the molecular mechanisms underlying invasion, a variety of in vitro assays have been developed. Most assays assess the interaction of tumor cells with the surrounding extracellular matrix (1 –4 ), although the invasive ability of other cell types could be measured. These methods involve cell invasion into a matrix coated on top of a filter. The layering of matrices requires precise thickness, because treatments are conventionally compared based on whether or not the cells have penetrated the matrix completely and appeared on the underside of the filter. In addition, until the development of methods using colorimetric, radiometric, or fluorescent dyes (5 –8 ), the counting of recovered cells from the lower chambers was slow and statistically uncertain (9 ). More recently an improvement in the invasion assay using a confocal laser scanning system to obtain images of light reflected by cells within the matrix has been described (10 ). In order to take full advantage of the ability of confocal laser scanning microscopy (CLSM) to examine specific fluorescence and to acquire images with high resolution, the technique presented here has modified the reflectance method by using propidium iodide (PI) and small pixel size to image nuclei in matrices on filters (11 –14 ).