Digital image Analysis: scratch assay


Counting cells


The scratch assay is a common method to study cell migration. A confluent cell monolayer is scratchwounded or scratched using a pipette tip or needle, introducing a cell-free area. Cells respond by closing the wound through cell proliferation and migration.

The migration of cells into the wound is monitored by capturing images at regular time intervals.

Scratch assays can be quantified by counting the cells migrated into the scratch.

The image shows wound healing of endothelial cells at 12 hours after the scratch was applied. The position of the initial scratch is indicated by the red lines.





Image analysis is realized by applying the standard set of filters and after thresholding the binary was analysed only in the region of the original sratch. Cells were counted as indicated and the result represent the cells migrated into the scratch with time.







Another method for scratch analysis uses clustering of regions covered with cells and analyse the ratio of cell-free to cell-covered area:


original images after analysis

t = 0

t = 0

t = 10 hours

t = 10 hours


The cell-free area accounts for 72% of the image when the scratch was applied. Cell migration into the scratch reduced the cell-free area to 45% after a period of 10 hours.