Optimized Scratch Assay for In Vitro Testing of Cell Migration with an Automated Optical Camera.

Published on Aug 8, 2018in Journal of Visualized Experiments1.163
· DOI :10.3791/57691
Michelle Vang Mouritzen4
Estimated H-index: 4
(RU: Roskilde University),
Håvard Jenssen40
Estimated H-index: 40
(RU: Roskilde University)
Sources
Abstract
Cell migration is an important process that influences many aspects of health, such as wound healing and cancer, and it is, therefore, crucial for developing methods to study the migration. The scratch assay has long been the most common in vitro method to test compounds with anti- and pro-migration properties because of its low cost and simple procedure. However, an often-reported problem of the assay is the accumulation of cells across the edge of the scratch. Furthermore, to obtain data from the assay, images of different exposures must be taken over a period of time at the exact same spot to compare the movements of the migration. Different analysis programs can be used to describe the scratch closure, but they are labor intensive, inaccurate, and forces cycles of temperature changes. In this study, we demonstrate an optimized method for testing the migration effect, e.g. with the naturally occurring proteins Human- and Bovine-Lactoferrin and their N-terminal peptide Lactoferricin on the epithelial cell line HaCaT. A crucial optimization is to wash and scratch in PBS, which eliminates the aforementioned accumulation of cells along the edge. This could be explained by the removal of cations, which have been shown to have an effect on keratinocyte cell-cell connection. To ensure true detection of migration, pre-treating with mitomycin C, a DNA synthesis inhibitor, was added to the protocol. Finally, we demonstrate the automated optical camera, which eliminates excessive temperature cycles, manual labor with scratch closure analysis, while improving on reproducibility and ensuring analysis of identical sections of the scratch over time.
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The work was funded by the Danish Council for Independent Research, Technology and Production, grant 4005‐00029; Strategic Project POCI‐01‐0145‐FEDER‐007440 funded by FEDER through Operational Programme Competitiveness Factors, the European Foundation for the Study of Diabetes (Novartis European Research Programme in Microvascular Complications of Diabetes), and FCT; and Pepper grant P30 AG028718 and NIGMS‐NIH P20GM109096 to EC.
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