Background

Cell migration plays an important role in both physiological and pathological processes ranging from embryonic development to angiogenesis and tumor metastasis (Le Clainche and Carlier, 2008). Cell motility is a highly orchestrated event that can be summarized as a cycle of four basic steps: i) membrane protrusion driven by actin polymerization, ii) stabilization of protrusion through integrin-mediated cell-matrix adhesion, iii) cell-body translocation driven by actomyosin contractile force, and finally, iv) rear release as a result of the mechanical action of contractile force and/or proteolysis of cell-matrix adhesion components (Sheetz et al., 1999; Ridley et al., 2003; Panetti et al., 2004; Stradal and Scita, 2006; Tomasevic et al., 2007; Le Clainche and Carlier, 2008). These processes involve dynamic remodeling of actin cytoskeleton which is dependent on de novo synthesis as well as regulation of important structural and regulatory components of actin cytoskeletal system.

Several methods have been developed to examine cell migration and can be separated into 2D and 3D assays. 2D migration assay have their advantages as they are typically easier and quicker to perform, however, lack the physiological representation that 3D assays may provide. We will provide a brief description of some of the other methods for cell migration. The scratch assay or wound healing assay is a commonly used technique to evaluate directed cell migration (Cory, 2011). In this assay, cells are plated to form a confluent monolayer and a stratch is introduced in the monolayer. The cell-free zone is then monitored as adjacent cells migrate to close the gap. While simple to perform, cell proliferation may bias the readout of this assay as that can influence scratch closure. In addition, cells in this assay are forced to move in one direction. The Boyden Chamber assay or transwell assay is another technique used to evaluate cell migration (Falasca et al., 2011). In this assay, cells are placed on one side of a porous membrane and allowed to migrate through the pores to the other side. An advantage of this assay is it allows for chemotaxis and works for both adherent and non-adherent cells. As with the scratch assay, migration in this assay is defined. Some disadvantages of this assay include difficulty to visualize cells and morphology due to transitive state of cells while migrating through pores.

In this protocol, we will describe the use of single cell migration assay to examine MDA-MB-231 cell migration. The key difference in this protocol is that we examine random cell migration rather than directed migration. This allows us to examine cell migration in a manner that is less influenced by cell-cell contact and its innate ability for directional persistence, i.e., ability to migrate randomly in a single direction. While obviously not a true physiological representation of in vivo cell migration, this assay allows us to quickly analyze effect of cellular manipulation on its ability to migrate. While this assay uses MDA-MB-231, this protocol can be performed with any migratory cell line. We have performed this assay using multiple cell lines with minor modifications (media used and amount of cells plated).