Scratch/Wound Healing Assay

Scratch/Wound Healing Assay2022-11-29T10:57:57-05:00


  • The scratch assay has been a staple method developed for the purposes of studying cell migration and proliferation patterns in vitro. This assay is an excellent method for characterizing and comparing the movement of cells under various controlled parameters.
  • The method was developed using the basic principle that a wound induction, or “scratch”, on a confluent cell monolayer will cause the migration of cells in an attempt to close the affected area. Analyzing the rate of wound closure in the plate/well over time can be used to make assumptions on how their in vivo cellular counterparts may behave under similar stimuli. [1]
  • The standard procedure includes plating the cells of choice in the growth medium to form a monolayer of cells. Once ideal confluency levels are reached, a scratching device is used to dislodge cells in a uniform manner at the center of the well. Images of the wound area are taken at the time points of choice to quantitatively analyze cell migration dynamics. [2]
  • A prime goal of this assay is to recapitulate in vivo cell processes and attempt to draw conclusions on migration dynamics in animate systems since much of the observed cell-cell interactions in the assay also happen within whole living tissue. For example, cell migration plays an integral role in processes such as the re-epithelialization of skin tissue or closing of the endothelium of wounded blood vessels. [3]
  • The area of the scratch is quantified using a custom-built Deep Learning image segmentation model trained on over 80k images.
  • Following image acquisition and analysis, the data can be used to make conclusions on a wide range of effects related to cell migration including the effects of cell-matrix on cell-cell interactions and can even be used to study the movement of individual cells.
  • This assay is frequently used because of its simplicity, low cost, and high throughput time.


InstrumentMolecular Devices ImageExpress Micro Confocal
Analysis MethodHigh content screening
Cell Model TypeAdherent monolayer
Cell Types AvailableHuman Lung Fibroblasts (NHLF), Human umbilical vein endothelial cells (HUVEC), intestinal epithelial cells (e.g., IEC-6, YAMC), other adherent cells, Client-provided cells. Custom cell-lines available on request
Test Article ConcentrationDose range based on expected EC50 of client compound
Number of Replicates3 replicates per concentration
Quality Controls0.5% DMSO (vehicle control)
Blebbistatin (positive control)
Test Article Requirements50 uL of 20 mM solution or equivalent amount of solid
Data DeliveryDose response curves, cell closure area and percent per concentration, evaluation of statistical significance respective to vehicle control

General Procedure

  1. Seed cells into a microwell plate at an appropriate pre-optimized seeding density on day 0.
  2. Apply “scratch wound” with Incucyte WoundMaker for controlled and consistent scratches, followed by gentle wash.
  3. Dose with desired drugs or compounds in serum starved media (to minimize background effects from proliferation).
  4. Image wells at 0 hour time points and at desired intervals (e.g. 4 hr, 8 hr, 12 hr, 24 hr, and 48 hr).
  5. Analyze images to quantify and compare wound closure across different treatment groups.
Fixed Scratch Assay Gif

Treatment of cells with blebbistatin significantly increased wound healing in fibroblasts over 48 hours. Scratch region highlighted in red.

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  1. Liang, C., Park, A. & Guan, J. In vitro scratch assay: a convenient and inexpensive method for analysis of cell migration in vitro. Nat Protoc 2, 329–333 (2007).
  2. Cory G. (2011) Scratch-Wound Assay. In: Wells C., Parsons M. (eds) Cell Migration. Methods in Molecular Biology (Methods and Protocols), vol 769. Humana Press.
  3. Vang Mouritzen, M., Jenssen, H. Optimized Scratch Assay for In Vitro Testing of Cell Migration with an Automated Optical Camera. J. Vis. Exp. (138), e57691, doi:10.3791/57691 (2018).