top of page
  • Writer's pictureNanoEntek

Wound / Scratch Assay Using Live-Cell Imaging System

Updated: Jan 2

Cell migration is a study of how cell moves within an organism, which is crucial for establishing and maintaining the proper organization of multicellular organisms such as proper immune response, wound healing, and detection of pathologies [1]. To study the movement of cells, a wound or scratch assay is a prominent technique widely used today.

Wound / Scratch Assay Basics

The wound or scratch assay involves making an artificial scratch in a cell monolayer to create an empty region then observe how cells migrate to fill in the cell-free gap over time. This assay is a simple and well-developed method for measuring cell migration in vitro.

Fig.1) Artificially made wound region being closed over time. Yellow marked region is where cells are located.


The method is straightforward: scratch a cell monolayer and compare images taken at regular intervals using time-lapse. The migration rate of the cells, as well as the movement of the cell population and individual cells, will be determined by this experiment [2]. The movement of cells can be measured by the confluence over time.

Confluence in cell culture indicates the area where cells are in contact and the how much of the vessel is covered by the cells [3]. Thus, as the cells migrate to close the scratch region, the level of confluence can be measured.

Wound Assay Method

1. Cell culture preparation

The wound assay begins with a cell culture in monolayer attached to the bottom of the vessel or plate. A scratch will be made and cells will be studied for further cell mobility and characteristics research.

Epithelial, fibroblastic, and smooth muscle cells are the cell types that are used most frequently.

Allow cells to proliferate and form a monolayer until adequate confluence is reached. Normally, the cell culture process takes a day or two.

2. Serum starvation (optional)

This is a commonly used condition in biology experiments to suppress the cellular activity such as cell proliferation in order to obtain more precise results. This step, however, could induce complex and unpredictable effects depending on cell types, hence it is optional [4][5].

3. Wound-making

In this step, a wound is made by scratching the monolayer of adherent cells with a sharp tool such as a pipette tip. Each scratch should be made uniformly to avoid width differences in each sample, especially when comparing multiple samples.

4. Preparation before incubation

After forming the scratch, gently wash the cell monolayer with culture media or phosphate-buffered saline (PBS). The media should then be replaced with new medium to give the nutrients required for cell migration.

5. Incubation

Place the cell sample in an incubator and allow cells to migrate over time. The time range could change depending on the cell type. Images collected at regular intervals using a live-cell imaging system allow for the complete process to be tracked from the start to the finish.

6. Analysis

The images obtained will then be analyzed using an analysis software.

This time-lapse video of a wound healing process was produced using JuLI Stage.

What can be analyzed using the wound/scratch assay?

The main goal of the wound assay is to monitor and analyze cell directionality and speed. This assay allows for a more thorough evaluation of cells by providing information on cell proliferation and interactions between cells in addition to cell migration. In pharmaceutical research and cosmetic industry, such wound or scratch assays are crucial for determining the effectiveness of ointments or skin-repairing treatments.


1.Trepat, Xavier, et al. “Cell migration.” Comprehensive Physiology, 2012, pp. 2369–2392,

2. Liang, Chun-Chi, et al. “In Vitro Scratch Assay: A Convenient and Inexpensive Method for Analysis of Cell Migration in Vitro.” Nature News, Nature Publishing Group, 1 Mar. 2007,

3.“Confluence.” Oxford Reference, Accessed 13 Sept. 2023.

4. Serum‐reduced Media Impacts on Cell Viability ... - Wiley Online Library, Accessed 13 Sept. 2023.

5. Martinotti, Simona, and Elia Ranzato. “Scratch wound healing assay.” Methods in Molecular Biology, 2019, pp. 225–229,

Objective lens

4x, 10x, 20x

Channel type

Brightfield, GFP, RFP, DAPI

Vessel type

Well plate, flask, dish, slide

Incubator installation


Recent Posts

See All


Commenting has been turned off.
bottom of page