Scratch Wound Migration and Invasion Assays for Live-Cell Analysis


Cell migration is a multistep process that is a fundamental component of many biological and pathological processes such as embryonic development, tissue re-organization, angiogenesis, immune cell trafficking, chronic inflammation, wound healing and tumor metastasis.

Cell migration is initiated by a stimulus that activates a set of signaling pathways leading to cellular polarization and a rapid reorganization of actin filaments and microtubules. Cells advance by protruding their membrane at their leading cell border, which is followed by dynamic substrate adhesion via integrin adherence to the substrate. Membrane retraction at the lagging cell edge finishes the cycle, which is then repeated in rapid succession. The summation of this process results in cell migration.

Cell invasion is one of the hallmarks of cancer. It is related to cell migration and plays a key role in metastasis. Metastasis is the leading reason for the resultant mortality of patients with cancer. The ability of tumor cells to form a metastatic tumor is primarily determined by the cell’s ability to change and reorganize its cellular morphology, and to degrade the extracellular matrix (ECM). Understanding the mechanisms concerned in tumor cell invasion may lead to limiting tumor progression and, as a result, to a reduction in mortality for many cancer patients.

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Cell Migration & Invasion Assay

Introducing Incucyte® Scratch Wound Cell Migration and Invasion&n...

An integrated solution enables real-time visualization and assessment of cell morphology in scratch wound assays (both label free and fluorescently labeled) up to six 96-well plates at once – all inside your tissue culture incubator.

With the Incucyte® Scratch Wound Assays, you can:

  • Assess the effect of treatments on full time course migration profiles.
  • Assess metastatic potential and define the effect of treatments on invasive phenotype.
  • Explore differential biology of cell migration and invasion in the same plate.

Visual validation of treatments and conditions in Incucyte® Scratch Wound Cell Migration and Invasion Assays. Differences in HT-1080 morphology and rate of wound closure between migrating and invading cell are observed. Rate of migration is greater than rate of invasion can also be observed. All 3 cell types migrate on collagen I coated wells. Highly invasive HT-1080 and MDA-MB-231 cell invade 3D collagen gel (1, 2 and 3 mg/ml). Relatively non-invasive MCF-7 cells do not invade 3D collagen gel under the condition of the assay.

Comparing Existing and Incucyte® Scratch Wound Cell Migration and Invasion Approaches

 Existing Scratch Wound AssayIncucyte® Scratch Wound Migration and Invasion Assay

Migration in the absence of chemotactic gradients


Low cell usage (<5,000 per well)


Suitable for non-adherent cells


Migration across a surface


Invasion through a 3D gel matrix


Integrated quantitation


Integrated cell observation


Precision, reproducibility


96-well throughput or higher


Kinetic read-out




Workflow: set up and walk away


Key Advantages

Key Advantages

  • Visualize cell migration and invasion, and assess morphological changes in real time with the scratch wound method
  • Assays are flexible, quantitative and easily reproducible with the Incucyte® Woundmaker tool
  • Monitor and quantify cell movements across a substrate (migration) or through a 3D gel matrix (invasion) in the same plate, up to six 96-well plates at once
  • Validated with a wide range of adherent primary, immortalized and tumor cell types

Visualize cell migration and invasion, and assess morphological changes in real time

  • Real time imaging and quantification of cell invasion through 3D biomatrix gels (e.g. Matrigel®)
  • Phase images allow the investigator to assess cell morphology at every time point
  • Graphical representation provides clear visualization of the initial wound and incremental wound closure at each time point

Figure 1. Quantifying cell migration and invasion with the scratch wound method. The green region denotes the scratch wound mask over time (t=0, t=2 hours) as HT-1080 cells migrate in to the wound region. The initial scratch wound mask, created immediately following wound creation, is shown in blue. Complete wound closure is observed at t=6 hour.

Reproducible quantitative measurement without labeling cells made easy...

The Woundmaker Tool is a 96-pin mechanical device designed to create homogeneous scratch wounds per plate in seconds at the press of a button. The unique tip design provides clean, consistent wounds without damaging the cells and creates a cell-free zone from a confluent monolayer of cells making the biology consistent and reproducible. It is a critical part of the scratch wound assays.

Figure 2a. HT-1080 monolayer scratch comparison using an Incucyte® Imagelock 96-W Plate. Note differences in width uniformity across wells, as well as scratch location within the wells from scratching manually using a 10 µL Pipette tip (top). The Woundmaker Tool accomplishes uniform scratches in all 96 wells with the push of a button (bottom). The time saving is tremendous.

View Incucyte® Scratch Wound Assay User Manual

Directly compare 2D migration and 3D invasion within an assay

  • Compare cell migration & invasion profiles and interrogate cell morphology changes by visualizing images and time-lapse movies
  • Pair wise analysis can be used to determine the specificity of pharmacological agents and the utility of potential drug targets

VIDEO: Investigate migration & invasion behavior in mixed cultures. The ability to image 2-color fluorescent scratch wound in addition to phase contrast now allows users to explore cell-cell interactions as it pertains to cell migration and invasion. Note the invasion of the HT-1080 cells, but not the MCF-7 cells into the wounded area.

Figure 2b.Pharmacological comparison of blebbistatin and GM6001 effect on the invasive phenotype of HT-1080 cells in Collagen I and Matrigel® matrix. Data show that blebbistatin inhibits invasion through both Collagen I and Matrigel® matrix (left), GM6001 inhibits invasion through Collagen I but not Matrigel® matrix (right).

Figure 2c. Scratch Wound Cell Migration quick guide.

Download the Incucyte® Scratch Wound Assay detailed protocol

Figure 2d. Scratch Wound Cell Invasion quick guide.

Amenable to a wide number of cell types

The Incucyte® Scratch Wound Assays are validated for over 20 different primary and immortalized cell types including HUVEC and tumor cell lines.


Scratch Wound Assays FAQs

Scratch-wound migration and invasion assays require loosely adherent cells that preferentially form monolayers. Cells that form strong adhesion to either the cell culture substrate or one another may form inadequate scratch wounds (incomplete removal of cells and cell debris from the length and width of the wound), inhomogeneous scratch wounds (different lengths, widths, and paths), or the cell monolayer may peel from the substrate during the scratch-wounding procedure. We recommend confirming the adhesion properties of your cells of choice on your substrate of choice by running a pilot experiment before choosing a cell type or cell line for use in a scratch-wound migration or invasion assay.

For details about forming reproducible scratch wounds across 24- and 96-well plates, check out the WoundMaker™.

Scratch-wound healing, or repair, is a common cell migration assay, measuring the speed and efficiency with which cells in a monolayer migrate to close a gap in the monolayer (a scratch wound). The length of time it takes to achieve scratch-wound repair is directly related to the cells, their ability to migrate on the substrate, and the width of the scratch wound. Depending on these factors, a scratch-wound assay may take anywhere from 4 hours to 24 hours, or longer. Since the assay requires frequent monitoring to assess progress – acquiring an image of each well’s repair progress every hour – an automated live-cell analysis system allowsyou to set up your scratch-wound assay, determine the imaging intervals, and walk away.

The scratch-wound migration assay is based on the formation of a scratch wound across a monolayer of cells and the assessment of how long it takes for the scratch wound to “heal”. Scratch wounds heal when cells on either side of the wound migrate into the empty space. This is a direct measure of cell migration along the surface of a solid, 2-D substrate. Cell-invasion assays are based on the scratch-wound approach, but include the addition of a gel matrix applied on top of the scratch wound. The scratch wound then heals, but it does so by the invasion of cells into and through the semisolid matrix. Some common matrices used for cell invasion assays include Collagen I and Matrigel®. The addition of the 3-D gel matrix requires the cells to penetrate and navigate (rather than simply migrating), which is not an ability shared by all cell types, but is common among tumor cell lines. In vitro-invasion assays, like the Matrigel® invasion assay, can closely recapitulate the biology of tumor cell invasion, in a plate-based format.

Scratch wounds are commonly generated with pins or cell scrapers, but these tools can lead to unreliable or inadequate scratch wounds. Variability in scratch wounds can make meaningful analysis difficult, as wound length and depth may be vastly different across a single plate.

The WoundMaker™ is specially designed to generate consistent results without damaging or deforming the plastic surface or ECM coating at the bottom of the well. The WoundMaker’s soft PTFE tips produce scratch wounds of a reliable width, without marring the cells’ paths. Scratched or dimpled plastic or ECM may impact the ability of migrating cells to cross the scratch wound, leading to anything from extended migration times to arrested migration. For optimal data quality and reproducibility, the WoundMaker™ is the best choice for safely creating scratch wounds in cell monolayers.

For more details on improving the reliability of your scratch-wound assays, check out our cell migration assay protocols at the bottom of this page.

Standard scratch wound and in vitro-invasion assays are typically set up in a tissue culture hood, moved to an environmentally controlled incubator, and removed to normoxic conditions and room temperature at regular intervals for microscopy. While most researchers are conscientious about keeping this excursion from incubation conditions as brief as possible, the actual time can stretch from 5 to 15 minutes, or longer. The change in atmospheric and thermostatic conditions can rapidly affect the cells’ growth and behavior,1-3 particularly when grown in small volumes, as is the case in 96well plates; smaller volumes of liquid are quicker to equilibrate to room temperature than larger volumes. Ensuring proper image registration between imaging series can be difficult when the plates are transported from the incubator to the microscope, and back again. Alternatively, experiments may be performed on microscopes with on-stage environmental enclosures. If leaks are encountered, then the environmental stability may be compromised and adversely affect cell health and motility. When you move your entire imaging apparatus into the incubator, as is now possible with the IncuCyte™ System, you’re able to image your cells actively behaving as they do under normal growth, migration, and invasion conditions. This means you’re more likely to capture accurate and meaningful data, from a morphological, physiological, and physico-chemical point of view. By keeping cells under optimal conditions throughout the assay, the cells are free to behave, without intrusion or interruption, yielding clearer, more meaningful data.

  1. Vergara, M., Becerra, S., Berrios, J., Osses, N., Reyes, J., Rodriguez, M., et al. Differential Effect of Culture Temperature and Specific Growth Rate on CHO Cell Behavior in Chemostat Culture. PLoS One. 9(4):e93865 (2014)
  2. Rezaei, M., Zarkesh-Esfahani, S.H., and Gharagozloo, M. The effect of different media composition and temperatures on the production of recombinant human growth hormone by CHO cells. Res. Pharm. Sci. 8(3):211-7 (2013)
  3. Mason, M., Sweeny, B., Cain, K., Stephens, P., and Sharfstein, S.T. Reduced Culture Temperature Differntially Affects Expression and Biophysical Properties of Monoclonal Antibody Variants. Antibodies. 3:253-71 (2014)

At first glance, that does seem like a tall order – tracking individual cells throughout the entirety of a cell-migration assay – but it doesn’t have to be a day-long ordeal, dashing between the incubator and the microscope! In-incubator, live-cell analysis platforms, like the Incucyte® Live-Cell Analysis System, can keep track of cell count, cell morphology, and cell migration, all while the cells remain in temperature- and atmosphere-controlled conditions. Live-cell analysis makes is possible to confirm the relative wound density of your scratch wound, the only direct measure of cell migration vs. cell proliferation in scratch-wound healing.

For additional details on the workflow and hands-on time, refer to the assay protocols (for scratch wound migration and scratch wound invasion) at the bottom of this page.

Ordering Information

Ordering Information



Cat. No.

Incucyte® Scratch Wound Analysis Software Module



Incucyte® Cell Migration Kit



Incucyte® Cell Invasion Accessories



Incucyte® Imagelock 96-well Plate (10 pack)



Incucyte® Imagelock 96-well Plate (50 pack)




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