Tools for Drug Discovery

The Scratch Wound Assay

Scratch wound assays are commonly used to assess the effects of drugs and drug candidates on the cellular proliferation and/or migration associated with wound closing. The IncuCyte™ is uniquely suited to the performance of this assay by providing ongoing, real-time images and quantitative data generated throughout the wound-closing process.

In the example above, HT-1080 cells were plated to confluence in a 24-well, Essen ImageLock™plate. A single scratch wound was created in each well using the Essen Woundmaker™. The plate was then inserted into the IncuCyte™, and the wound area within each well was repeatedly imaged at fixed time intervals.

Graphical Representation of Wound Closing over Time in the Presence of Cytochalasin-D

Wound confluence can be graphed over time to quantitatively evaluate the characteristics of wound closing in the presence of pharmacological agents.

Phase_Contrast Imaging Facilitates Evaluation of Cellular Morphology

To the right, HT-1080 cells are displayed 24-hours post-wounding. Cells in the upper box were not treated with any drug. Cells in the lower box were treated at wounding with 5μM Paclitaxel. In addition to the wound failing to close, cellular morphology is clearly aberrant.

The Essen Woundmaker™

One of the most important aspects of performing the scratch wound assay is generating consistent, good quality wounds. Essen Instruments has developed a hand-operated Woundmaker™ that fulfills exactly this requirement.

The 24-well, Essen ImageLock™ plate is inserted into the plate holder of the Essen Woundmaker™. Four tips are attached to a hand-held lever that moves the tips into and out of the wells. The Woundmaker™ generates one wound per well, 4 wells at a time, one column at a time. Wounds are straight and of high quality. Wound locations and widths are very consistent within each plate and from one plate to the next.

The Woundmaker™ is designed to be used in combination with the Essen ImageLock™ plates. These plates facilitate very precise, repeated imaging within the wound area. The Woundmaker™ and ImageLock™ plates are synchronized so that the wound will always occur in the appropriate location within each plate. Both of these products are essential for the IncuCyte™ scratch wound software to function properly.

How the Data are Generated

The IncuCyte™ scratch wound software works by generating a "wound mask" for each well. An initial wound mask is created for the very first image (at time 0) that clearly delineates the border of the wound. A revised mask is generated for each subsequent image to track wound closure. Both the phase contrast images and the wound mask overlays can be viewed through the IncuCyte™ software. This process is represented below:

Titration of Paclitaxel

Wound closure can be automatically monitored using one or both of two distinct metrics: wound confluence (% confluence) and wound width (μm). In the example below, titration is represented graphically both in terms of wound confluence (left) and wound width (right). Note that as wound confluence increases over time, wound width decreases.

Paclitaxel exhibits a highly potent effect on wound closing, with significant inhibition even at the low concentration of 320pM.

Titration of Y27632 - Wound Confluence

Titration of Y27632 - Wound Width

Unlike Cytochalasin-D and Paclitaxel, Y27632 shows little effect on the rate of wound closure.

Conclusion:

Essen Instruments introduces a novel product package to enhance the performance of the scratch wound assay:

1. The IncuCyte™
2. The IncuCyte™ scratch wound assay software
3. The Essen Woundmaker™
4. The Essen ImageLock™