INTRODUCTIONThein vitro scratch ass

INTRODUCTION
Thein vitro scratch assay is a straightforward and economical method to study cell migrationin vitro 1
. This method is based on the observation that, upon creation of a new artificial gap, so called
‘‘scratch’’, on a confluent cell monolayer, the cells on the edge of the newly created gap will move toward the opening to close the ‘‘scratch’’ until new cell–cell contacts are established again. The basic steps involve creation of a ‘‘scratch’’ on monolayer cells,capture of images at the beginning and regular intervals during cell migration to close the scratch, and comparison of the images to determine the rate of cell migration.
One of the major advantages of this simple method is that it mimics to some extent migration of cellsin vivo.Forexample,removal of part of the endothelium in the blood vessels will induce
migration of endothelial cells (ECs) into the denuded area to close the wound
2
. Furthermore, the patterns of migration either as loosely connected population (e.g., fibroblasts) or as sheets of cells (e.g., epithelial and ECs) also mimic the behavior of these
cells during migrationin vivo. Another advantage of the in vitro scratch assay is its particular suitability to study the regulation of cell migration by cell interaction with extracellular matrix (ECM)
and cell–cell interactions. In other popular methods such as Boyden chamber assays, preparation of cells in suspension before the assays disrupts cell–cell and cell–ECM interactions. In addition,the in vitro scratch assay is also compatible with microscopy including live cell imaging, allowing analysis of intracellular signaling events (e.g., by visualization of green fluorescent protein (GFP)-tagged proteins for subcellular localization or fluorescent resonance
energy transfer for protein–protein interactions) during cell migration. On the other hand, it is also probably the simplest method to study cell migration in vitro and only uses the common and inexpensive supplies found in most laboratories capable of cell culturing.
Although it is developed and more suitable for measuring migration of population of cells, thein vitro scratch assay has also been combined with other techniques, such as microinjection
or gene transfection, to assess the effects of expression of exogenous
genes on migration of individual cells
3–5
. The migration path of
individual cells in the leading edge of the scratch is tracked with the
aid of time-lapse microscopy and image analysis software. Capturing of an image in the beginning of the experiment with fluorescence microscopy can mark the cells with expression of exogenous
gene or downregulation of endogenous genes by RNA interference (e.g., using a GFP marker). By comparing the tracks of these cells with surrounding control cells under the same experimental conditions allows determination of the role of a particular gene in the regulation of directional cell migration using the assay.