<em>In vitro</em> Cell Migration and Invasion Assays

Abstract

Migration is a key property of live cells and critical for normal development, immune response, and disease processes such as cancer metastasis and inflammation. Methods to examine cell migration are very useful and important for a wide range of biomedical research such as cancer biology, immunology, vascular biology, cell biology and developmental biology. Here we use tumor cell migration and invasion as an example and describe two related assays to illustrate the commonly used, easily accessible methods to measure these processes. The first method is the cell culture wound closure assay in which a scratch is generated on a confluent cell monolayer. The speed of wound closure and cell migration can be quantified by taking snapshot pictures with a regular inverted microscope at several time intervals. More detailed cell migratory behavior can be documented using the time-lapse microscopy system. The second method described in this paper is the transwell cell migration and invasion assay that measures the capacity of cell motility and invasiveness toward a chemo-attractant gradient. It is our goal to describe these methods in a highly accessible manner so that the procedures can be successfully performed in research laboratories even just with basic cell biology setup. Video LinkThe video component of this article can be found at

Optimization of the Wound Scratch Assay to Detect Changes in Murine Mesenchymal Stromal Cell Migration After Damage by Soluble Cigarette Smoke Extract

“…However, traditionally the scratch assay has lacked a standardized set-up and quantification protocol, which has led to issues with reproducibility 7,10 . Many of the modifications and optimizations for improving the scratch assay have reduced these issues, including individual live-cell imaging, standardized stopper-based assays, and three dimensional micro-pillar assays 4,8,10 . However, these assays can be difficult to customize or adapt 10 and for some applications, these techniques can also be cost prohibitive.…”

“…In contrast, too many cells in the monolayer (100% confluent, or stacked cells) may well result in contact inhibition, and therefore change the migratory characteristics of the cells. Some variations of the scratch assay protocol recommend cell growth to 100% confluence and do not consider this potential limitation for certain contact-sensitive cells such as MSCs 8 . The same concerns should be applied to the degree of damage that is inflicted on the cells (Step 1.5).…”

“…To assess both cellular senescence and cell proliferation, a combination of senescence-associated beta-galactosidase and BrDU or EdU incorporation can be performed on the cells at the end of the assay Optimizations presented in this protocol address some of the limitations present in previous work done to standardize scratch assays. Whereas scratch assay protocols traditionally take scratch width measurements from a snapshot of the scratch 8,9 , the protocol presented here uses area measurements from images taken along the entire length of the scratch to increase statistical rigor and to take into consideration inconsistencies in the width of the initial scratch. Based on repeated testing (data not shown), scratch width cannot be assumed to be perfectly consistent throughout its length.…”

“…This technique assumes that the cell population is relatively homogenous, and that this will result in relatively homogenous migration characteristics. Depending on the application, it may be more relevant to consider the migration of individual cells instead 8 . Moreover, the scratch assay protocol described here is dependent on a population of cells that will not proliferate rapidly enough to confound analysis of migration over a 4-7 hr period.…”

“…The assay entails seeding the designated cells, growing them to complete or near confluence, and scratching the resultant monolayer with a sterile needle or pipet tip 6 . Analysis is most commonly performed by comparing the width of the scratch over multiple time points at randomly selected locations [7][8][9] . Despite its prominent use, the scratch assay is confounded by issues with reproducibility and quantification.…”