Confocal assay for invasion: use of propidium iodide fluorescence and laser reflectance to quantify the rate of migration of cells through a matrix.

Background: Most assays used to measure invasion are based on manual counting of the number of cells that have migrated completely through commercial coated filters. We describe here a confocal fluorescence-imaging method that can assess the relative rates of invasion of cells into a matrix.

Methods: After being seeded on the matrix and a period of incubation, the cells are fixed and treated with RNase. Propidium iodide is then added to stain the double-stranded DNA. A confocal microscope system is used to obtain high-resolution images of the red propidium iodide fluorescence and laser reflectance from optical sections at increasing depths in the matrix. The section with high laser reflectance marks the top of the matrix.

Results: Data were calculated as the total area of red fluorescence above background in each section and were plotted as a percentage of the summed fluorescent areas in all sections.

Conclusions: Because the distance into the matrix of the nuclei can be calculated by measuring from the reflective upper surface of the matrix, the method is useful for assessing the rate of cell migration and for comparing the ability of different cells to invade through different matrices under varying conditions.