Wound healing assay

Introduction to wound healing assays

Wound healing assay or scratch assay is a commonly used method to study collective cell migration in vitro. This method is based on the observation that, upon creation of an artificial gap (‘scratch’) on a confluent cell monolayer, the cells on the edge of the scratch will migrate towards the opening and establish new cell-cell connections, hence closing the gap. The initial steps of a wound healing assay involve the creation of a scratch in a cell monolayer, subsequent capture of images at multiple time points to monitor the progression of gap closure, and quantification of the percentage of gap closure and/or cell migration rate1.


Researchers can perform wound healing experiments with these CytoSMART devices: full-plate scanner Omni, fluorescence cell imager Lux3 FL, mini live-cell imagers Lux2 and Lux3 BR.


Wound closure analysis using live-cell imaging

Using the capabilities of the plate imager, CytoSMART Omni, users can investigate the migration patterns of cells in multi-well plates and compare multiple treatments/conditions. Here, a wound healing assay was performed to study the migration of C6 rat glioma cells. The CytoSMART Omni allows users to visualize the progression of the gap closure, providing an overview of multiple wells (Figure 1A) as well as zooming in on selected wells or areas of the scratch (Figure 1A; Figure 2)). The integrated image analysis algorithm automatically highlights (in blue; Figure 1A) the areas of the wells lacking cells; it also quantifies the gap surface closure (Figure 1B) and cell migration speed (Figure 1C).

Wound healing diagram

Figure 1 | The analysis of C6 glioma cells migration. C6 rat glioma cells were plated in a 48-well plate, wounded with a pipette tip, and monitored for 17 hours using the CytoSMART Omni. (A) The integrated image analysis algorithm automatically selects the cell-free (‘scratch’) areas (highlighted in blue) and monitors the progression of the scratch closure over a specified period of time. The generated output provides information on the (B) area of the scratch over time and (C) cell migration speed. (B) and (C) display the data of the gap closure of the four wells shown in (A).

Figure 2 | Time-lapse video of C6 glioma cells migration and scratch closure. The monolayer of C6 rat glioma cells, plated in one of the wells of a 48-well plate, was wounded with a pipette tip and recorded via a whole-well overview using the CytoSMART Omni. The brightfield images were acquired every hour for a total period of 23 hours.

The CytoSMART Omni: efficient comparison of samples

Another example highlights the advantage of using the CytoSMART Omni for high-throughput comparison of multiple test samples. In the video below, NIH-3T3 mouse fibroblasts were seeded in a single 96-well plate and treated with a range of concentrations of a myosin II inhibitor, blebbistatin, and an inhibitor of actin polymerization, cytochalasin D. Next, the cells were subjected to in vitro scratch assay. The images were captured using the CytoSMART Omni for 48 hours with a 1-hour imaging interval. The results show that increasing concentrations of blebbistatin and cytochalasin D lead to inhibition of NIH-3T3 cell migration (Figure 3).

Figure 3 | Analysis of NIH-3T3 cell migration following the treatment with blebbistatin and cytochalasin D. The monolayers of NIH-3T3 mouse fibroblasts were treated with blebbistatin and cytochalasin D, wounded with a pipette tip, and monitored for 48 hours (1-hour imaging interval) using the CytoSMART Omni. The areas in blue define the parts of the wells lacking cells (‘scratch’). White-purple arrows indicate the increasing concentrations of the added compounds. Scale bar = 1000 µm.

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