The cell-edge protrusion assay has been shown to directly correlate with cell migration. Therefore it can be used as a preliminary method for identifying critical proteins and signaling mechanisms involved in cell motility. The method is fast, simple, cost effective and does not require fluorescent labeling or using an expensive fluorescent microscope.
Demonstrating the procedure will be Michal Gendler, a student from my laboratory. Add two milliliters of one normal hydrochloric acid solution at the center of a glass bottom dish and incubate for 20 minutes at room temperature. Wash the dish three times with two milliliters of PBS.
Dilute fibronectin at 10 micrograms per milliliter concentration in PBS and add 200 microliters of the diluted solution to the glass center dish. Incubate for one hour at 37 degrees Celsius. Prepare 1%BSA solution and PBS and pass it through a 0.2 micrometer filter.
Denature the solution by incubating at 70 degrees Celsius for 30 minutes in a pre-warmed water bath. Wash the coated glass bottom dish with two milliliters of PBS three times. Add two milliliters of denatured BSA solution and incubate the dish at 37 degree Celsius for one hour.
Wash the glass dish with two milliliters of PBS three times. Before 16 to 18 hours of experiment at 0.7 million cells per 10 centimeter diameter tissue culture plate to achieve 70 to 80%confluency. The next day, add two milliliters of trypsin solution to the tissue culture plate and incubate for two to three minutes until the cells get detached.
Add five milliliters of the medium to inactivate the trypsin. Using a hemacytometer, count the cells and plate 20, 000 cells in two milliliters of the complete medium in a bottom dish. Then incubate the dish with plated cells in an incubator for 15 minutes.
Turn on the heating unit and set it at 37 degrees Celsius one hour before imaging. Also, turn on the carbon dioxide unit and set it at 5%10 minutes before imaging. Switch on the microscope and camera.
Turn on the computer and open the microscope acquisition software. Set the magnification on a 40X dry lens phase contrast. Set the total movie duration to 10 minutes with a time interval of five seconds.
After 15 minutes of incubation, place the glass bottom dish with adhered cells into an adapter and fix it. Insert the adapter with the dish into the slots in the microscope stage. Take off the dish cover, place the carbon dioxide lid and open the carbon dioxide valve.
Locate an appropriate cell for imaging. Start movie acquisition after focusing on the cell. To perform the image analysis, select the straight tool in the image analysis software and make eight lines of 20 arbitrary units perpendicular to the protrusions in a radial arrangement at every 45 degrees, including lamella and cell-edge.
In the main toolbar, go to image, select Stacks and then click on Reslice to generate a kymograph picture describing the movement of single points within the cell membrane. Using the kymograph images, extract and manually count the number of protrusions, retractions and ruffles in each of the eight regions in the cell marked by the grid lines. These numbers represent the frequency of protrusions, retractions and ruffles per 10 minutes.
Determine the protrusion persistence, distance and velocity by kymography analysis. To determine the protrusion distance, draw perpendicular line from the base of the protrusion to the highest peak of the protrusion. Press M to measure the length of the line in pixels and ensure that the pixel to micrometer ratio is known to convert the length in micrometers.
Quantification of protrusions, reactions, and ruffles was performed manually per 10 minutes and the average frequency obtained for protrusions and retractions was 5.1 and 2.1 for ruffles. The representative kymograph had a protrusion distance of approximately 4.8 micrometers and a protrusion time of approximately 0.6 minutes. An example of a cell that should be excluded from the analysis is represented.
The kymography analysis revealed that the cell was not present in its spreading phase and did not show any clear membrane protrusions. The most important thing is to choose correct cells for imaging. A proper cell should be in its spreading phase and should not touch other cells to avoid cell-cell communication and signaling.
The method can be used as a preliminary tool for testing cytoskeletal dynamics involving cell motility before deciding to perform more results demanding cell migration assays.
