How-To Video: Scratch Assay App

This How-To Video provides a quick overview on setting up and running BioTek’s Scratch Assay application. The app is designed to analyze plates that have been processed using BioTek’s Autoscratch wound making tool to monitor cell migration over time and generate quantitative data characterizing the wound healing process.


Hello and welcome to BioTek’s Scratch Assay App. This app is designed to analyze plates that have been processed using BioTek’s Autoscratch wound making tool. It allows you to automatically collect multiple data points, monitor cell migration over time, and generate quantitative data to characterize the wound healing process.

Upon opening the Scratch Assay App, a new blank scratch assay template is generated. To navigate to previously generated data or to import previous settings, click on the menu icon in the upper left corner.

To set up a new experiment, let’s step through the workflow using the menu on the left. Here you’ll specify the incubation temperature, either 37 degrees or room temp, the total duration up to 72 hours, your measurement interval, and the Corning plate type you are using, either 96 or 24 wells. Under plate setup we’ll select the imaging area of interest, you can image your whole plate or just a few columns it’s up to you, and you can also assign well information to distinguish control wells from any drug treated wells.

Now we’ll go ahead and setup the auto-exposure. Select one to four representative wells and click Start Auto Exposure. After giving your file a name, you’ll be prompted to place your plate on the carrier. The auto expose process serves as a visual check to monitor cell health and scratch quality before moving ahead with the experiment. If everything looks good, click start run.

Now that our wound healing experiment is complete, let’s set up the analysis. Click Add to choose representative wells to determine appropriate settings. I’d recommend selecting an early time point before wound healing has begun as well as a later time point where the wound is nearly closed. This will ensure that settings are applicable across all time points and treatments. A mask is applied to distinguish cells from background. The stringency of the mask is controlled by adjusting the threshold value. While the default settings are usually suitable you may need to fine tune and optimize. For example, a threshold value that is too low will fail to exclude your background. While a threshold value that is too high will start to exclude some of the dimmer cell regions.

You can adjust the minimum object size to help eliminate any small debris that is being incorrectly identified or to address any fragmentation that you see within the cell layers. Let’s look at another well to make the settings are optimal. These results look good. You can apply brightness and contrast adjustments, and while this won’t have any effect on the analysis it can enhance the appearance of the images and movies. Also note that the current mask view will be included in the final images. Choose the display ON, OFF, or filled in that you prefer and then choose Apply to all wells to apply your analysis settings across all of the wells and all of the time points. This can take a moment depending on the total number of images.

Once the analysis is complete, graphs are automatically generated to show the change in average wound width or wound confluence over time. It will also calculate the maximum rate of wound migration. You can export your individual images or movies. Select the wells that you’d like to export and time points of interest. Now you can choose to export images in order to generate individual image files for each well, or for movies adjust the frame rate and format and then choose export movie. You can also export your wound width and confluence curves. These can be exported individually or combined and overlaid on a single graph. Individual graphs can be exported here under export curves while the combined curves can be configured here. Select the wells you’d like to combine and preview by clicking display. The generated graph can then be exported.

All of the raw data and well assignments are also available. And be exported to a spreadsheet by choosing export data.

This application provides a simple and straightforward way to collect and visualize your data from wound healing experiments. If you have any questions or support needs please contact our Technical Assistance Center.