This protocol provides a new method for an old technique, and it will provide a new approach for an large number of basic science researchers. The main advantages of this technique are it's cost effective nature as well as its adaptability to be used by a wider scientific audience. To prepare a migration plate, use a light colored permanent marker to draw a line down the center of the back of each well of a 48 well plate and to draw three hash marks to divide each well into three separate areas of interest.
Then plate 1.5 to two times 10 to the fourth single passage cardiac fibroblasts into each well and culture the cells under normal culture conditions for 24 to 48 hours until they reach 90 to 95%confluency. When the cells are ready, remove the supernatant from each well and use a sterile P200 pipette tip to make a one pass scratch along the drawn lines. Rinse the wells with low serum medium to remove any unattached cardiac fibroblasts and add 500 microliters of low serum medium to each well.
If pharmacological agents are being used, add the agents to the appropriate wells. Next, use an inverted microscope with a 20x objective to capture two zero hour images per well using the markings to position each well to allow imaging of the top half of the scratched line. Then move the plate to position the bottom half of the scratched line into view to collect the second image and place the plate in the cell culture incubator for 24 hours.
At the end of the incubation, wash each well with non-sterile PVS and add 500 microliters of 4%paraformaldehyde to each well in a fume hood. After 10 minutes at room temperature, wash each well three times in fresh PBS for five minutes per wash. After the last wash, permeabilize the cells with 300 microliters of permeabilizing solution per well and gentle rocking for 30 minutes at room temperature.
At the end of the incubation, replace the permeabilizing solution with 1%Kumasi brilliant blue stain for a 10 minute incubation with gentle rocking at room temperature. Followed by three, five minute washes in PBS with rocking. After the last wash, add 300 microliters of PBS to each well and carefully position the plate in the same position as for the zero hour image before capturing two 24 hour images per well as demonstrated.
At the end of the experiment, open the images and an appropriate imaging analysis software program and create a new layer on the zero hour image. Double click to change the name of the new layer line layer and select the brush tool. Change the pixel size to 10 and the color to red and draw two separate lines to outline the scratch.
The lines should not touch any cells. Open the 24 hour image in the imaging software and select the move tool. Holding down the control button, click both the lines and the background layers, click in the center of the zero hour image and drag both layers to the middle of the 24 hour image.
In the 24 hour image, click the zero hour background layer and change the opacity to 50%Holding the control button, click both the zero hour background and line layers and click edit and free transform to free transform the layers Using free transformation, align the zero hour background and line images to the 24 hour background image so that the area migration lines are positioned correctly in the 24 hour image. When the line layer has been successfully overlaid, right click to delete the zero hour background layer. The remaining line layer can be used to determine the number of cells that migrated into the scratch area over 24 hours.
Then save the new migration image as both a Photoshop and a TIFF or JPEG file. To quantify the number of migrating cells, open the migration image and a program that accepts TIFF or JPEG files and manually count the number of cells located in between and touching the two red lines for both images for each well. Then record the number of migrating cells per image.
To determine the area of migration, open the 24 hour image that contains the lines of migration in the imaging software and save the image as migration area image. After saving, click the background layer and right click to delete the layer. Use the brush tool to fill in the area between the scratch lines, with the same color that was used for the lines and click image, mode, and gray scale to change the image to black and white.
Save the image and open the migration area image and an appropriate image analysis software program. Click edit, and invert. To determine the area of the line of migration, click image, adjust, and threshold.
The black migration area will turn red and the percent area will be indicated in the threshold box. Then record the percent area for the line of migration and confirm that this value is paired with the number of migrating cells for the same image. In this analysis, 46 fibroblasts from non-diabetic hearts and 129 fibroblasts from diabetic hearts migrated during the 24 hour experimental time period.
Measurement of the migration area as demonstrated, revealed that the non-diabetic scratch area made up 24.78%of the total area measured, while the diabetic migration scratch area made up 16.77%of the total area measured. Normalizing to the area of migration provides a better and more rigorous assessment of cell migration and negates potential human error. For example, if only the number of migrated fibroblasts are compared, it would appear in this analysis that the number of cells that migrated in this well was twice that of the migrated cells in the second well.
When the area of the scratch was used to normalize the data, however, the ratio of the fibroblasts to the migration area was observed to be nearly the same in both wells. Be sure to recapture the area of migration in the 24 hour image, as you captured in the zero hour image. Following this procedure, immunohistochemistry can be conducted to examine protein expression within the cells.
We believe this newer approach to the scratch migration assay will allow for more avenues of scientific research to open up that previously were not available.
