Optimization of the Wound Scratch Assay to Detect Changes in Murine Mesenchymal Stromal Cell Migration After Damage by Soluble Cigarette Smoke Extract

The overall goal of this assay is to detect changes in the migratory capacity of lung mesenchymal stromal cells after damage with soluble cigarette smoke extract. This method can be used to answer key questions in the field of lung regenerative biology, including how migratory capacity of lung progenitor cells can be improved to aid in lung repair. The main advantage of this technique is that it's been optimized to provide better quantitative measurements for cell migration while still remaining economical to perform and easy to customize us.

In this demonstration, the scratch assay will be used to quantify the migratory capacity of murine lung mesenchymal stromal cells, or MSCs. After exposure to soluble cigarette smoke extract, the MSCs are grown in complete media on 60 millimeter plates and damaged as described in the protocol text. An appropriate confluence is important for the scratch assay because it ensures that the imaging software can correctly identify the boundaries of the scratch.

This example shows MSCs at 95%confluence appropriate for scratching in a sterile environment. Remove the lid from a plate of damaged MSCs and lay a straight edge, such as a sterilized plastic ruler across the top rim of the plate. A straight edge minimizes the amount of rotational adjustments needed for the plate when it is later placed on the microscope stage for imaging.

Without removing the media, use a sterile 200 microliter pipette tip guided by the sterile straight edge to make four parallel scratches across the plate, approximately five millimeters apart and 50 millimeters long. Generating multiple scratches on a 60 millimeter plate does not alter the microenvironment in a way that affects cell migration for MSCs, but may be a consideration for other cell types. Aspirate the media and gently wash the plate with two milliliters of prewarm complete media.

This prevents cells from adhering to the center of the scratch and removes cells that have been loosened by the scratching. Replace with four milliliters of fresh complete media using a sterile, permanent marker label each scratch from one to four on the underside of the plate at locations that will not interfere with imaging. The scratches initial images of each scratch must be taken because it is important that images from the same scratch are compared before and after migration, since there can be variation in scratch width.

If the scratch assay is performed on multiple plates, it is recommended that initial images of all the scratches on a single plate are taken before scratches are made on the next plate. Use an inverted phase contrast microscope with a camera to acquire the images needed for analysis. Position the culture plate on the microscope and use the low power objective to locate scratch.

Number one. It is imperative that the scratch appears completely horizontal on the screen while keeping the scratch completely horizontal and in the center of the field of view, obtain as many images as necessary to encompass 90%of the scratch's length. Obtain images from distinct portions of the scratch with no overlap between images.

Do not take images from the first and last 5%of the scratch's length because the refraction of light at the edges of the culture dish over exposes the images, making them impossible to analyze. Save images from scratch, number one in a folder. Obtain images for scratches two, three, and four in the same way, and save images from each scratch in a separate folder.

Return the plates to the incubator immediately after this initial imaging. Let cells migrate for four to seven hours prior to starting this assay. Download the Image J software and install the wound healing tool plugin.

Open the image file in image J, then click process and find edges to highlight the cells surrounding the scratch for the wound healing tool. To calculate the scratch area, click image adjust and color threshold, and in the dropdown menu labeled threshold color, change the value to BNW within the color threshold menu. Move the bottom slider completely to the right to make the image completely black, and then adjust the top slider to generate maximum contrast between the scratch and cell front.

Slowly adjust the top brightness slider from left to right until the image cleanly displays the contour of the scratch. Once the edges of the scratch have been identified, click more tools. In the image J toolbar select MRI wound healing tool from the dropdown menu and press the M button to highlight the scratch area and output the computed area.

In a results pop-out window, copy and paste all numbers from the results window into an Excel spreadsheet. Make sure to separate data from individual scratches. After the cells have migrated for four to seven hours, repeat the image acquisition procedure for the scratches image the plates in the same order they were scratched so that cells on each plate have had equal time to migrate.

The cells can be imaged at multiple time points depending on their sensitivity to both temperature and pH changes. The final scratch images are then analyzed using Image J in the same way as demonstrated for the initial scratch images. Scratch areas can also be calculated to quantify migration, correct.

Confluence is critical for the success of the scratch assay. A successful scratch of murine lung MSCs at 95%confluence is shown here. In contrast, a 70%confluence is too low for a successful scratch and can produce false boundaries.

These are representative pre-migration and post-migration original images with corresponding scratch area boundaries as defined by the imaging software. These graphs show the average calculated scratch area in pixels for each of the four scratches made across a plate of murine lung MSCs at 95%confluence either before or after migration with exposure to either 0%or 4%cigarette smoke extract. A comparison of the calculated average change in scratch area revealed less migration of murine lung MSC after exposure to 4%cigarette smoke extract.

While attempting this procedure, it is important to remember to optimize and carefully monitor cell confluence for the particular cell type we were working with and practice generating the scratches to ensure they're as uniform as possible. Following this procedure, other techniques like immunohistochemistry can be performed to answer additional questions about alterations and protein expression that may affect cell migration.