Optimized Scratch Assay for <em>In Vitro</em> Testing of Cell Migration with an Automated Optical Camera

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.

Immunomodulatory potential of Nisin A with application in wound healing

“…The wells were washed with 250 μl PBS, and a scratch was manually performed with a 200 μl pipet tip, during the second wash. After removing the PBS, Nisin A was added in a concentration of 25 μg/mL in media. The plate was incubated at 37 °C and in 5% CO 2 for 24 hours in the automated optical camera system oCelloScope (BioSense, Denmark), where migration of the cells was captured every hour and processed by the UniExplorer software (version 8.0.0.7144 (RL3)), as previously demonstrated by our group . For the HUVEC cells, the precoating was done previously to seeding the cells.…”

“…The plate was incubated at 37 C and in 5% CO 2 for 24 hours in the automated optical camera system oCelloScope (BioSense, Denmark), where migration of the cells was captured every hour and processed by the UniExplorer software (version 8.0.0.7144 (RL3)), as previously demonstrated by our group. 17,18 For the HUVEC cells, the precoating was done previously to seeding the cells. Cells treated with free amino acids in the same mole-ratio as in Nisin A were used as a negative control and epithelial growth factor (EGF) was used as a positive control at 500 ng/ml.…”

Aggregation of platelets, proliferation of endothelial cells and motility of cancer cells are mediated by the B?1(15)-42 residue of fibrin(ogen)

“…The scratch wound-healing assay was applied as previously described [30 ]. It is a widely used approach for estimating cell migration, and is especially useful for the study of migration of cancer cells [31]. Briefly, 4×10 5 cells were seeded into 24-well cell culture plates and allowed to grow to form a confluent monolayer (24 h).…”

Neurotensin, substance P, and insulin enhance cell migration

“…The plate was incubated in an oCelloScope (BioSense Solutions ApS, Denmark) in a conventional incubator (37°C and 5% CO 2 ) for 24 hours, where migration was monitored. The imaging and measurements of cell migration were automatically performed by the Uniexplorer software (version 8.0.0.7144 (RL3)) of the oCelloScope, capturing 40 pictures per scan area every hour during a 24‐hour period, as previously described …”

“…The imaging and measurements of cell migration were automatically performed by the Uniexplorer software (version 8.0.0.7144 (RL3)) of the oCelloScope, capturing 40 pictures per scan area every hour during a 24-hour period, as previously described. 28 After 24 hours, the cells were harvested using TRI reagent (Sigma-Aldrich, T9424) for further use (qPCR). The cell migration and proliferation into the scratch was calculated as the percentage of closure from the initial scratch.…”