A Quantitative, Facile, and High-Throughput Image-Based Cell Migration Method Is a Robust Alternative to the Scratch Assay

Abstract

Cell migration is a key phenotype for a number of therapeutically important biological responses, including angiogenesis. A commonly used method to assess cell migration is the scratch assay, which measures the movement of cells into a wound made by physically scoring a confluent cell monolayer to create an area devoid of cells. Although this method has been adequate for qualitative characterization of migration inhibitors, it does not provide the highly reproducible results required for quantitative compound structure-activity relationship evaluation because of the inconsistent size and placement of the wound area within the microplate well. The Oris™ Cell Migration Assay presents a superior alternative to the scratch assay, permitting formation of precisely placed and homogeneously sized cell-free areas into which migration can occur without releasing factors from wounded or dead cells or damaging the underlying extracellular matrix. Herein the authors compare results from the scratch and Oris™ cell migration assays using an endothelial progenitor cell line and the Src kinase inhibitor dasatinib. They find that using the Acumen™ Explorer laser microplate cytometer in combination with the Oris™ Cell Migration Assay plate provides a robust, efficient, and cost-effective cell migration assay exhibiting excellent signal to noise, plate uniformity, and statistical validation metrics.

Sensitive and Accurate Quantification of Human Leukocyte Migration Using High-Content Discovery-1 Imaging System and ATPlite Assay

“…Both Discovery-1 or ATPlite assays would be applicable for the study of less abundant leukocyte subsets (e.g., antigen-presenting cells or regulatory T cells) or rare sources of cells extracted from human tissue (e.g., human brain, skin, tumors, or lymph nodes) where yields are often very low. Previous studies have developed HTS methods for investigating the migration of adherent cells during scratch/wound-healing models 16,17 or extravasation of cells through tissue. 18 However, these are quite distinct from the methods and applications we have developed here.…”

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

“…Cell migration is highly coordinated and vital to many physiological processes such as tissue development, repair, and regeneration, as well as pathological processes such as cancer metastasis and arteriosclerosis 1 . An understanding of cell migration is particularly relevant to emerging therapies used to repair damaged tissues and treat pathological conditions, including cell transplantation technologies and artificial tissue grafting 2 .…”

“…Assays are frequently conducted over 7 to 12 hr, however for cell lines displaying slower migration and longer assay times, proliferation becomes a confounding variable 7,10 . Lastly, senescent cells generated by the scratching process can release factors that interfere with the extracellular signaling required to close the gap in the monolayer 1 . Optimizing the scratch assay requires creation of a consistent gap that does not interfere with surface properties, minimizing assay time length, and preventing unwanted cell death during the manipulation.…”

“…Importantly, such an assay must be sufficiently sensitive to detect relatively subtle changes in cellular migratory capacity after damage. Current methods for quantifying cell migration, including scratch assays, trans-well migration assays (Boyden chambers), micropillar arrays, and cell exclusion zone assays possess a range of limitations in reproducibility, customizability, quantification, and cost-effectiveness 1,4,5 . The optimized scratch assay described here demonstrates robust outcomes, quantifiable and image-based analysis capabilities, cost-effectiveness, and adaptability to other applications.…”

Investigating the Reproducibility of In Vitro Cell Biology Assays Using Mathematical Models

“…(a) (b) t = 0h t = 0h Although in vitro cell biology assays are popular, problems associated with reproducibility have been reported in previous studies [35,120]. Variables such as experimental equipment and environment can make reproducibility difficult [35,58,122].…”